bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 189: The Zinc-finger Protein 148(zfp148) Modulates Smooth Muscle Marker Genes via Interaction With Nf-1 in Vascular Disease
Authors: Salmon Morgan , Wu Andrew , Shankman Laura S , Greene Elizabeth , Zehner Zendra E , Merchant Juanita L , Owens Gary K , Upchurch Gilbert R , Ailawadi Gorav
Published: March 2022

Objective:
We and others have shown that smooth muscle (SMC) marker gene down-regulation is an early event in vascular disease and involves the transcription factor KLF4. However, the molecular mechanisms mediating continued repression following KLF4 release remain unknown. Recently, we have shown that ZFP148 is critical in SMCs during aortic aneurysm formation; however, no studies have investigated the molecular mechanisms of this process. The study objective was to determine what factors mediate SMC down-regulation after KLF4 binding is diminished.

Methods and Results:
8-12 week male mice (n=8 per group) underwent carotid ligation and were harvested at day 7 for chromatin immunoprecipitation analysis (ChIP) for KLF4, KLF2, ZFP148, NF-1, Sp1, Sp3 and YY1. ZFP148, NF-1 and KLF2 were significantly elevated following ligation injury. In separate experiments, 8-12 week male (n=10/group) C57/B6 mice underwent carotid ligation and were harvested at 1, 3, 7, and 14, days for ChIP analysis for ZFP148 and NF-1. ZFP148 and NF-1 bound SMC marker genes SM-actin, SM22α and SM-MHC 7 and 14 days following injury. Since ZFP148 and NF-1 bound and could mediate SMC marker gene repression and ZFP148 has no current known role in SMC down-regulation, 8-12 week male (n=14/group) ZFP flx/flx Myh11 Cre+(SMC specific ZFP148 KO), Myh11 ZFP148 flx/wt Cre+(Heterozygous ZFP148 KO) and Myh11 ZFP wt/wt Cre+ (WT) underwent carotid ligation following tamoxifen administration. Neointimal area was significantly increased 21 days following injury in ZFP148 SMC KO mice. In separate experiments, ZFP148 flx/flx ERT Cre+ (ZFP148 KO, n=12/group) and ZFP148 flx/flx ERT Cre-(WT) mice also underwent carotid ligation following tamoxifen treatment. Neointimal area was also significantly increased in ZFP148 KO mice. ChIP assays were performed in vitro and in vivo using NF-1 KO cells. ZFP148 bound via ChIP analysis to SMC marker genes in vitro and in vivo; however, ZFP148 binding declined significantly in NF-1 KO SMCs versus WT SMCs.

Conclusions:
ZFP148 activity is critical for vascular disease and helps modulate smooth muscle phenotypic modulation during vascular injury in coordination with NF-1. As such, ZFP148 is another zinc finger protein with potential therapeutic relevance.
Abstract 194: Inhibition of Epigenetic Reader BRD4 Mitigates Intimal Hyperplasia Following Angioplasty in Rat Carotid Arteries
Authors: Wang Bowen , Takayama Toshio , Zhang Mengxue , Shi Xudong , Roenneburg Drew , Kent K. Craig , Guo Lian-Wang
Published: March 2022

Background:
Intimal hyperplasia (IH) causes recurrent lumen narrowing leading to failure of revascularization. BRD4 represents an emerging class of epigenetic “readers” and has recently been shown to play a crucial role in cancer. While blockage of BRD4 activity with a specific inhibitor (JQ1) rescues heart failure in a mouse model, the role of BRD4 in vascular diseases is not known.

Hypothesis:
BRD4 stimulates IH by promoting a transformation of vascular smooth muscle cells (SMCs) from quiescent to proliferative and migratory phenotypes.

Methods and Results:
Balloon angioplasty was performed to induce IH in rat carotid arteries. Immunohistochemistry showed that 7 days after balloon injury, 85% of cells in the neointima were BRD4 positive, in contrast this ratio was only 7% in the media of uninjured control (n=3 animals). A pronounced increase of BRD4 was also observed in restenotic versus normal human veins. Blocking BRD4 with periadventitially applied JQ1 (500 μg/rat) reduced IH by 75% and SMC proliferation (PCNA staining) by 60% compared to vehicle control (n=4, p<0.05) at 14 days post injury. Consistently, in vitro treatment with 1 μM JQ1 markedly inhibited PDGF-BB induced proliferation (by 72%) and migration (by 63%) in rat aortic SMCs as well as proliferation in human aortic SMCs (by 56%). Whereas PDGF receptor (PDGFRα) was found to be drastically up-regulated in restenotic versus normal human veins, it was reduced by 55% in JQ1-treated rat carotid arteries compared to vehicle control 14 days after balloon injury (n=4, p<0.05). Accordantly, Western blotting showed a 70% decrease of PDGFRα in cultured rat SMCs 48h after treatment with 1 μM JQ1.

Conclusions:
Our study is the first to demonstrate that inhibiting epigenetic reader BRD4 mitigates IH, likely by down-regulating PDGFRα expression. This epigenetic regulation may provide novel targets for effective therapeutic prevention of IH.
Abstract 195: Endothelin Converting Enzyme-1 (ECE1) is Essential in Adult Normal Physiology
Authors: Kristianto Jasmin , Johnson Michael , Radcliff Abigail , Patel Forum , Zastrow Ryley , Jill Koch , Wang Xiaohu , Yuan Baozhi , Blank Robert
Published: March 2022

Endothelin converting enzyme-1 (ECE1) catalyzes the conversion of inactive big endothelin 1 (ET1) to active ET1. Homozygous
Ece1
knock out (KO) mice die
in utero
or at birth, displaying multiple abnormalities including mandibular hypoplasia and cardiac outflow tract malformations, in spite of the presence of ample tissue ET1. In contrast, increased ECE1 activity and circulating and/or tissue ET1 are associated with many adult cardiovascular diseases, including idiopathic pulmonary fibrosis (IPF), a chronic and fatal lung disease. There is an apparent paradox between the need for ET1 in development and its harmful effects in adult disease. Therefore, our lab developed a conditional
Ece1
KO mouse, in which the gene is ablated following tamoxifen (tam) treatment. We hypothesized that ECE1 serves to localize ET1 signals to specific cell populations and is essential in adult normal physiology. We studied the following groups: mice given vehicle rather than tam, mice lacking tam-inducible Cre recombinase, mice harboring a normal
Ece1
allele (

Ece1
+/flox

), and the experimental animals (Cre

Ece1
-/flox

). Mice were treated with vehicle or tam at 8-9 weeks of age. Cre

Ece1
-/flox

mice showed 85-100% mRNA knock-down efficiency 8 weeks after tam treatment, but there was no significant difference in ET1 mRNA level. By 17 weeks of age, Cre

Ece1
-/flox

mice have significantly reduced body weight compared to controls (p=0.005) and most also have impaired breathing. Two weeks after treatment, Cre

Ece1
-/flox

mice had lower blood pressure relative to controls, which persisted until euthanasia at 17-20 weeks old (p=0.004). Between 17-20 weeks of age, most of Cre

Ece1
-/flox

mice develop pectus excavatum, cardiac arrhythmia, reduced cardiac output, and depleted adipose tissue mass. Histological examination revealed eosinophilic crystalline pneumonia and increased collagen in the lung. These findings are consistent with development of IPF in the experimental mice. Our findings show that
Ece1
ablation in post-natal animal results in a severe progressive disease requiring euthanasia for humane considerations, suggesting that ectopic activation of ET1 by other tissue proteases is the primary mechanism underlying the association of increased ET1 signaling in disease states.
Abstract 21: B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis
Authors: Morris-Rosenfeld Sam , Perry Heather M , Srikakulapu Prasad , McSkimming Chantel , Gonen Ayelet , Prohaska Thomas A , Tsimikas Sotirios , Witztum Joseph L , Bender Timothy P , Taylor Angela , McNamara Coleen A
Published: March 2022

Rationale:
B cells contribute to atherosclerosis through subset specific mechanisms. Whereas some controversy exists about the role of B-2 B cells, B-1a B cells are atheroprotective. The function of B-1b B cells, a unique subset of B-1 cells that can produce T cell-independent (TI) memory, has not been studied within the context of atherosclerosis.

Objective:
To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet induced atherosclerosis.

Methods and Results:
We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation specific epitopes (OSE) on LDL in vitro in response to activating stimulation. Additionally, we demonstrate that B-1b cells provide direct atheroprotection after adoptive transfer into B and T cell deficient (Rag1-/-Apoe-/-) hosts. To support these findings, we utilize a B cell specific Id3 knockout mouse model (Id3BKO), which develops increased numbers of B-1b cells systemically, to demonstrate that these mice develop attenuated atherosclerosis and increased OSE IgM antibodies compared to wild-type controls (Id3WT) after being fed a Western diet for 16 weeks. Finally, we report that the presence of a functionally relevant homozygous SNP in ID3 in humans associates with increased proportion of circulating B-1 cells and anti-MDA-LDL IgM and that the percentage of circulating B-1 cells is directly associated with the amount of anti-MDA-LDL IgM suggesting clinical relevance.

Conclusions:
These results provide novel evidence that B-1b B cells produce atheroprotective OSE IgM antibodies and protect against atherosclerosis in mice, and suggest that similar mechanisms may occur in humans.
Abstract 218: Dynamics of Blood Leukocytes Following ST Segment Elevation Myocardial Infarction
Authors: Yuan Jinqing , Chu Fei , Yao Yi , Xu Jingjing , Wang Miao
Published: March 2022

Blood leukocyte counts are linked to cardiovascular risk stratification, and excess activation of neutrophil and monocytes has been observed in patients of myocardial infarction (MI). However, the profile of major subsets of leukocyte following ST Segment Elevation MI (STEMI) is still unclear. It was aimed to compare relative change of neutrophil, monocytes, T and B lymphocytes in blood leukocytes in patients of STEMI undergoing percutaneous coronary intervention (PCI). STEMI patients subject to PCI were consecutively recruited. Peripheral blood was collected upon patient admission to hospital (referred to as T0), one day(T1), and/or four days (T4) after PCI, and were measured by flow cytometry (n=12, 18, 34 for T0, T1 and T4, respectively). Compared to T0, percentage of neutrophil (CD45+CD66b+) decreased, whereas T cell (CD3+CD45+) increased, at both T1 and T4. Percentage of monocytes (CD14+CD45+) and B cells (CD19+CD45+) both increased at T4 relative to T0. Interestingly, monocyte- and neutrophil- expression of CD11c, an inducible integrin alpha subunit, increased significantly at T4 compared to T1, despite a dichotomous change in population percentage for these two cell types. A dynamic shift in different leukocyte subsets, as well as in their activation status, occurs in STEMI patients underwent PCI. Such changes might reflect different aspects of biology in patients of STEMI with concurrent standard therapy, and might inform selection of new markers for cardiovascular risk stratification.
Abstract 220: Deficiency in Triggering Receptor Expressed in Myeloid Cells-1 Substantially Attenuates Atherosclerotic Lesion Development
Authors: Zysset Daniel , Weber Benjamin , Rihs Silvia , Freigang Stefan , Banz Yara , Saurer Leslie , Mueller Christoph
Published: March 2022

TREM-1 is a potent amplifier of pro-inflammatory responses in myeloid cells. While the significance of TREM-1 in acute microbial-induced septic shock has long been acknowledged, our previous studies in experimental models of colitis have also suggested an important role for TREM-1 in driving non-infectious and chronic inflammatory diseases.

Here, we aim to define the role of TREM-1 in atherosclerosis, which is recognized as a chronic inflammatory disorder of the arteries with a major involvement of monocytes and macrophages. TREM-1-deficient (Trem1-/-) mice were backcrossed with apolipoprotein E-deficient (Apoe-/-) mice and were fed a high cholesterol/fat diet for 16 weeks. Compared with Trem1+/+ Apoe-/- mice, Trem1-/- Apoe-/- mice were substantially protected from diet-induced atherosclerosis. Interestingly, while the number of individual atherosclerotic plaques did not differ between the two groups of mice, the average lesion size and total surface area of atherosclerotic lesions were significantly reduced in Trem1-/- Apoe-/- mice. Moreover, analysis of early atherosclerotic lesions revealed no difference between the two groups. Hence, TREM-1 appears to be involved in the progression, rather than the initiation of atherosclerotic lesions. In line with the reduced lesion size, FACS analysis of aortic cell preparations revealed substantially lower frequencies of infiltrating CD11b+ myeloid cells in Trem1-/- Apoe-/- mice. To gain insight into the underlying mechanisms of TREM-1-mediated effects in atherogenesis, we are currently analyzing mRNA expression profiles of aortic segments derived from Trem1+/+ Apoe-/- and Trem1-/- Apoe-/- mice. These studies will be complemented by immunohistochemistry stainings and by in vitro studies that are aimed to directly determine the impact of TREM-1 on foam cell generation and cholesterol efflux.
Abstract 226: Neuronal Nitric Oxide Synthase Maintains Endothelial NO Release after Inhibition of NADPH Oxidase 4
Authors: Brunssen Coy , Eickholt Claudia , Langbein Heike , Langbein Heike , Brux Melanie , Goettsch Claudia , Goettsch Winfried , Morawietz Henning
Published: March 2022

The NADPH oxidase isoform NOX4 mainly produces H
2
O
2
. Previously we could show, that NOX4 is the major endothelial NOX isoform and constitutively active. Regulation of NOX4 and formation of reactive oxygen species is involved in NO release. The transcription factor NRF2 is a key mediator of cellular adaptation to redox stress. Therefore, regulation of NOX4 on the transcriptional level by NRF2 might be directly linked to NO release and endothelial function. Besides endothelial nitric oxide synthase (eNOS), a role of neuronal nitric oxide synthase (nNOS) in endothelial NO and H
2
O
2
release has been proposed. In this study, endothelial cells (HUVEC) were constantly exposed to high laminar shear stress (24 h, 30 dyn/cm°). Application of shear stress stimulated NO formation and induced elongation of endothelial cells in the direction of flow. Lentiviral overexpression of NOX4 strongly increased H
2
O
2
release, while downregulation using shNOX4 decreased H
2
O
2
release. Furthermore, application of shear stress caused downregulation of NOX4 as well as upregulation of eNOS and antioxidative response via NRF2 and its target genes NQO-1 and HMOX-1. H
2
O
2
can increase NO release by eNOS. We could show that downregulation of NOX4 leads to upregulation of eNOS mRNA and protein expression accompanied by attenuation of NRF2 pathway under flow conditions. Determination of NO release confirmed these results. This supports a compensatory mechanism maintaining a stable NO release after NOX4 inhibition in response to flow. Attenuation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2 and its target genes. In addition, shNRF2 enhanced the shear stress-dependent downregulation of NOX4. Finally, we detected a stable mRNA and protein expression of nNOS in endothelial cells, unaffected by flow. Interestingly, downregulation of NOX4 as well as of NRF2 resulted in strong induction of nNOS expression.

In conclusion, our data suggest an important role of H
2
O
2
production by NADPH oxidase 4 in maintaining NO release and endothelial function in response to flow. Inactivation of NADPH oxidase 4 attenuates NRF2 pathway and increases neuronal nitric oxide synthase as compensatory mechanisms.
Abstract 229: Collagen Inhibitory Peptide R1R2 Mediates Vascular Remodeling by Decreasing Inflammation and Smooth Muscle Cell Activation
Authors: Chiang Hou-Yu
Published: March 2022

The extracellular matrix (ECM) is a major constituent of the vessel wall. In addition to providing a structural scaffold, the ECM controls numerous cellular functions in both physiologic and pathologic settings. Vascular remodeling occurs after injury and is characterized by endothelial cell activation, inflammatory cell infiltration, phenotypic modulation of smooth muscle cells (SMCs), and augmented deposition of collagen-rich ECM. R1R2, a peptide derived from the bacterial adhesin SFS, with sequence homology to collagen, is known to inhibit collagen type I deposition in vitro by inhibiting the binding of fibronectin to collagen. However, the inhibitory effects of R1R2 during vascular remodeling have not been explored. We periadventitially delivered R1R2 to carotid arteries using pluronic gel in a vascular remodeling mouse model induced by blood flow cessation, and evaluated its effects on intima-media thickening, ECM deposition, SMC activation, and inflammatory cell infiltration. Morphometric analysis demonstrated that R1R2 reduced intima-media thickening compared to the control groups. R1R2 treatment also decreased collagen type I deposition in the vessel wall, and maintained SMC in the contractile phenotype. Interestingly, R1R2 dramatically reduced inflammatory cell infiltration into the vessel by ~78%. This decrease was accompanied by decreased VCAM-1 and ICAM-1 expression. Our in vitro studies revealed that R1R2 attenuated SMC proliferation and migration, and also decreased monocyte adhesion and transendothelial migration through endothelial cells. Together, these data suggest that R1R2 attenuates vascular remodeling responses by decreasing inflammation and by modulating SMC proliferation and migration, and suggest that the R1R2 peptide may have therapeutic potential in treating occlusive vascular diseases.
Abstract 232: E-selectin-targeting Microparticle-mediated Delivery of MicroRNAs for the Treatment of Atherosclerosis
Authors: Ma Shuangtao , Tian Xiao Yu , Mu Chaofeng , Shen Haifa , Bismuth Jean , Wong Wing Tak
Published: March 2022

Rationale:
E-selectin on inflamed endothelium promotes atherosclerosis and also provides binding sites for targeted drug delivery. We identified an E-selectin-targeting thioaptamer (ESTA) and conjugated it onto a multistage vector (MSV) to form an ESTA-MSV microparticle. In this study, we tested targeted delivery of therapeutic microRNAs (miRs) loaded in the ESTA-MSV for the treatment of atherosclerosis.

Methods and Results:
The expression of E-selectin was up-regulated, while the expressions of miR-146a and miR-181b were down-regulated, in TNF-α-treated human microvascular endothelial cells (HMVECs). MiR-146a and miR-181b mimics were packaged into ESTA-MSV microparticles. We found that both the miR-146a and miR-181b significantly attenuated TNF-α-induced up-regulation of adhesion molecules. Moreover, the monocyte adhesion to HMVECs was also attenuated by the miR-146a or miR-181b. The ApoE
-/-
mice were injected with ESTA-MSV-packaged miR-146a or miR-181b mimic biweekly through tail vein for 12 weeks. Injection of miR mimics resulted in a 4.7- and 5.7-fold overexpression of miR-146a and miR-181b in mouse aortas. The immunofluorescence staining showed the co-localization of E-selectin and miRs in aortic endothelium. MiR-146a and miR-181b significantly inhibited atherosclerotic lesion size, lipids deposition, macrophage and lymphocyte infiltration in plaques, and gene expression of adhesion molecules in aortas.

Conclusions:
E-selectin-targeting delivery of miR-146a and miR-181b inhibits atherosclerosis. These findings provide a novel delivery system for miR-based therapy of atherosclerosis.
Abstract 234: Ramp2 Is a Novel Flow-sensitive Gene Both In vitro and in vivo
Authors: Ni Chih-Wen , Kim Chan Woo , Jang Inhwan , Jo Hanjoong , Lawson Nathan D
Published: March 2022

Ramp2 is a member of ramps (receptor activity modified proteins) family, which comprises a single transmembrane domain and involves in protein transport. Specifically, ramp2 is required to transport calcitonin-receptor-like receptor (CRLR) to the plasma membrane, which can function as an adrenomedullin receptor. In addition, ramp2 significantly expresses in endothelial cells but the function of ramp2 in endothelium in response to blood flow has not been reported yet. Here, we first report the expression of ramp2 is significantly altered by different flow conditions. In cultured HUVECs, ramp2 mRNA level decreases over 90% (p<0.01) in oscillatory shear (OS) flow compared to laminar shear (LS). Similarly, ramp2 is down-regulated (reduced 79%, p<0.01) under low and oscillatory shear condition in vivo when performing partial carotid ligation in mouse model. Moreover, a very similar result was obtained from deep sequencing data acquired in zebrafish. Zebrafish embryos were left untreated or injected with tnnt2 morpholinos at one-cell stage that in turn prevents heart beating and stops blood flow. We developed a method to acquire endothelial-enriched mRNAs from these embryos at 2 days post fertilization (dpf), and then samples were subjected to deep sequencing analysis. The data showed that ramp2 decreases 60% (p<0.01) at no flow condition, which is consistent with the observation in mouse and cultured human cells. Together, these data suggests that ramp2 is a novel flow-sensitive gene despite the detail function and significance is still to be determined.
Abstract 237: Role of Runx2 in Vascular Responses to Angiotensin II-Induced Hypertension
Authors: Ribeiro Mauricio , Smith Leslie A , Thalji Nassir M , Roos Carolyn M , Miller Jordan D
Published: March 2022

Increases in local angiotensin II (ANGII) signaling have been implicated in a number of diseases, including atherosclerosis, hypertension, and aneurysm. Endothelial dysfunction is strongly associated with increased ANGII signaling and the pathogenesis of these diseases. While increases in Runx2 have been implicated in cardiovascular calcification and vascular stiffening in diabetes, little is known regarding the role of Runx2 in the regulation of endothelial function. In the present study, we hypothesized that Runx2 is a key mediator of impaired vascular function and increased vascular wall stiffness provoked by ANGII. Wild-type mice (WT) and littermates that were deficient in one copy of Runx2 (HET) were implanted with osmotic minipumps containing saline or a pressor dose of ANGII (1000 ng/kg/min). Blood pressure was measured non-invasively using tail-cuff methods (CODA) and mice were sacrificed following 14 days of treatment. Changes in endothelium-dependent relaxation (relaxation to acetylcholine, R
ACH
) and passive pressure-diameter relationships were measured in vitro (Living Systems arteriograph). In WT mice, treatment with ANGII for 14 days resulted in profound impairment in R
ACH
(85 ± 4% vs 47 ± 14% p<0.05) and slight reductions in vascular diameter at high distending pressures (~10% reduction in maximal diameter). While Runx2 deficiency did not alter R
ACH
or vascular distensibility/compliance in saline-treated mice, loss of one copy of Runx2 in ANGII-treated mice dramatically improved R
ACH
compared to their wild-type, ANGII-treated littermates. Interestingly, impairments in the pressure-diameter relationship with ANGII infusion were not affected by loss of one copy of Runx2. Critically, reductions in Runx2 did not significantly alter the pressor response to ANGII. In conclusion, our data suggest that Runx2 may play an integral role in the pathogenesis of ANGII-induced endothelial dysfunction, with these changes occurring independently of alterations in extracellular matrix remodeling and vascular stiffness. These data suggest that Runx2 may play a key, context-dependent role mediating vascular responses to ANGII.
Abstract 251: Selective Deletion of Smad4 in Smooth Muscle Cells Causes Thoracic Aortic Aneurysm in Mice
Authors: Carnevale Daniela , Da Ros Francesco , Carnevale Raimondo , Cifelli Giuseppe , Bizzotto Dario , Braghetta Paola , Bressan Giorgio , Lembo Giuseppe
Published: March 2022

TGFβ signaling have been implicated in the onset of thoracic aortic aneurysms (TAA). Recently, it has been described that mutations in SMAD4, an intracellular transducer required in the TGFβ canonical pathway, present aortic disease in humans. Although transgenic models recapitulating mutations of TGFβ pathway have been studied, Smad4 role in the onset of TAA has never been explored.

Smad4 homozygotes mice are embryonically lethal, thus, we postnatally targeted the gene, using a tamoxifen (TAM)-inducible Cre recombinase under the smooth muscle myosin heavy chain promoter. Activation by TAM suppressed Smad4 in aorta SMCs of Smadf/f;Myh11-CreERT2 mice. We found that, most of these mice died 4/6 months later from hemothorax due to rupture of thoracic aorta, while untreated mice survived. Echocardiography showed a progressive dilation of aorta and TAA formation. Histology revealed progressive fragmentation of elastic lamellae with increased inflammatory infiltrates at sites of disarrangement, rich in macrophages. At the molecular level, we found that lack of Smad4 was followed by increased levels of PSmad2 and PSmad1/5/8 early after TAM, suggesting an enhancement of TGFβ receptors activity, a condition that could increase non-canonical TGFβ signaling, the canonical one being depressed. We actually found a significant increase in one of the non-canonical TGFβ signaling, i.e. the p65 subunit of NFkB, a protein complex controlling DNA transcription and involved in regulating responses to different stress stimuli, including cytokines. Given the particular relevance that the latter molecules may have in TAA, we tested expression of some of them, finding a selective IL-1β upregulation, produced and stored in cells as an inactive precursor and released in its active form by caspase-1 cleavage. In turn, caspase-1 requires NLRP3 inflammasome, a multiprotein platform that assembles in response to infection and tissue damage and is responsible for activation of inflammatory caspases. Therefore, we also tested NLRP3 mRNA levels and found them to be significantly upregulated.

We demonstrated for the first time that selective Smad4 deletion in the SMC of adult mice is sufficient to induce ultrastructural damage and immune reaction culminating in the onset of TAA.
Abstract 252: Elastin-derived Peptides Induce M1 Macrophage Polarization Promoting Abdominal Aortic Aneurysm Formation
Authors: Dale Matthew A , Xiong Wanfen , Carson Jeffrey S , Ruhlman Melissa K , Baxter B T
Published: March 2022

Objective:
Abdominal aortic aneurysm (AAA) is a disease characterized by inflammatory cell infiltration and extracellular matrix (ECM) degradation. Damage to the ECM results in release of elastin-derived peptides (EDPs). EDPs recruit inflammatory cells and may influence macrophage polarization. BA4, a monoclonal antibody that specifically recognizes a six peptide sequence in EDPs, can block the EDP-mediated effect on macrophages. Our hypothesis is that EDPs induce M1 differentiation and that blocking the EDP-mediated M1 differentiation of macrophages will reduce aneurysm progression.

Methods:
Mice were given weekly intraperitoneal injections of BA4 (10 mg/kg) or IgG (10 mg/kg) after aneurysm induction via the calcium chloride (CaCl2) induced aneurysm model. Aortic tissue was then removed and subjected to Western blot, gelatin zymography, and histological analysis at various time points (3 days, 1, 2, 4, and 6 weeks) after aneurysm induction. Bone marrow-derived macrophages (BMMs) were isolated and treated with various doses of EDPs and their gene expression profiles were analyzed by qPCR. Additionally, BMMs were treated with IFN-γ to induce a pro-inflammatory M1 phenotype or IL-4 to induce an anti-inflammatory M2 phenotype. M1 or M2 BMMs were given intravenously to mice, which then underwent aneurysm induction.

Results:
BA4 treatment significantly reduced aortic dilation, ECM degradation, and macrophage infiltration. EDP treatment of naïve macrophages induced a response similar to IFN-γ resulting in M1 activation as seen by expression of M1 associated markers such as TNF-α and IL-1β. Additionally, administration of M1 polarized BMMs to CaCl2-treated mice significantly increased aortic dilation compared to administration of M2 polarized BMMs.

Conclusions:
EDPs cause a pro-inflammatory M1 response similar to that seen with IFN-γ treatment. Reducing the M1 response by inhibition of EDP-mediated signaling or administration of M2 polarized BMMs reduces aneurysm formation. Targeting the macrophage phenotype is a potential therapeutic target for AAA.
Abstract 253: Vibrational Photoacoustic Imaging of Lipid in Murine Abdominal Aortic Aneurysms and Atherosclerosis
Authors: Sangha Gurneet S , Phillips Evan H , Li Rui , Cheng Ji-Xin , Goergen Craig J
Published: March 2022

Vibrational Photoacoustic Tomography (VPAT) is a novel imaging modality that uses pulsed laser light to generate an acoustic signal, making it ideal to obtain structural and compositional information. These characteristics allow detection of lipid distribution in diseased aortae by overlaying ultrasound (40 MHz transducer) and VPAT lipid signal (Fig. 1A). We used VPAT to test the hypothesis that arterial remodeling of suprarenal AAAs cause less lipid accumulation in the false lumen compared to other regions prone to atherosclerosis. To induce AAAs, osmotic minipumps were subcutaneously implanted in the backs of apolipoprotein E-deficient mice (n=4; 25±4.7 weeks old) to deliver 1000 ng/kg/min angiotensin II for 28 days. Aortae were imaged in situ, 21 days after AAA diagnosis, using VPAT at 1100 nm for blood (Fig. 1B) and 1210 nm for lipid (Fig. 1C) in a long-axis view. We quantified lipid and blood signal in the false lumen and the aortic wall proximal and distal to the AAAs. We observed a two-fold increase in lipid signal distal to the AAA (p<0.05) and small increase in lipid signal proximal to the AAA (p>0.05), likely due to atherosclerosis in these regions (AAA: 34.1±8.4 AU; distal: 68.5±19.4 AU; proximal: 56.7±9.7 AU). Blood signal was relatively constant along the vessel (AAA: 43.5±17.4 AU; distal: 40.2±28.2 AU; proximal: 27.8±17 AU). The aortae of these animals are prone to atherosclerosis, consistent with VPAT images and previous histological data. Interestingly, our findings show that while blood signal did not vary with location, lipid signal was reduced in the false lumen of these dissecting AAAs compared to other regions. These results suggest that VPAT may be useful to quantify atherosclerosis development, a disease becoming more prevalent with the current increase in obesity and diabetes.
Abstract 254: Sirtuin-1 Activation Has no Effect on Aortic Root Dilatation in Marfan Mice
Authors: Hibender Stijntje , ter Braake Anique , 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. MFS 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. Losartan has been shown to inhibit aortic root dilatation in MFS patients. The beneficial effect of losartan suggests the detrimental involvement of angiotensin II receptor 1 (AT1R) signaling as a cause for aortic pathology. Angiotensin II (Ang-II) can induce cellular senescence, and downregulates sirtuin-1 (SIRT-1) expression. SIRT-1 is an NAD-dependent deacetylase which can prevent senescence. Among other functions, resveratrol can activate SIRT-1 and is effective in inhibiting aortic root dilatation in MFS mice. To further delineate the underlying mechanism, SIRT-1 activator SRT1720 is examined as therapeutic in MFS mice.

Methodology:
In the current study we investigated the effect of SRT1720 on aortic root dilatation in the Fbn1 C1039G/+ MFS mouse model. Eight week old mice were treated for two months with SRT1720. Aortic senescence and aortic root diameters were measured.

Results:
Effectiveness of the compound was established by measurement of body weight and granulocyte blood count. As expected, SRT1720-treated MFS mice showed decreased weight gain and increased granulocyte counts, confirming that SRT1720 was effective. While aortic senescence was decreased in the SRT1720-treated mice, this had no beneficial effect on aortic root dilatation.

Conclusions:
While SRT1720 prevented aortic senescence, this did not have an effect on aortic root dilatation in MFS mice. Thus the beneficial effect of resveratrol on aortic root dilatation can not be explained via its function as SIRT-1 activator.
Abstract 264: Circulating Tnni3k Levels Are Useful Diagnosis Biomarker for Acute Ischemic Myocardial Diseases
Authors: Lai Zhong-fang
Published: March 2022

Our recent data showed that TNNI3K, a MAP kinase specifically expressed in cardiac muscles and interacted with cardiac troponin I, TNNI3K high-expression enhanced cardiac performance, attenuated ischemia-induced ventricular remodeling in mice with myocardial infarction, and protected the myocardium from ischemic injury.

In the present study, availability of circulating TNNI3K level was investigated using anti-TNNI3K polyclonal antibodies with ELISA, in patients diagnosed as AMI, chronic heart failure and acute renal failure; and in healthy volunteers groups. Data shown that circulating TNNI3K levels were significantly higher in AMI when compared with other two groups (p<0.001), indicating that measurement of circulating TNNI3K is a novel and useful diagnosing tool for AMI. Then, we made an anti-TNNI3K monoclonal antibody to investigate whether circulating TNNI3K levels play the same role for diagnosis of ischemic myocardial diseases, the availability of serum TNNI3K level was investigated with anti-TNNI3K monoclonal-antibodies in AMI, chronic heart failure and acute renal failure patients, and in healthy volunteers respectively. Data shown that circulating TNNI3K levels were significantly higher in AMI (p<0.001) and sensitivity and specificity are higher, ROC curve is more best than that of polyclonal antibodies, indicating that measurement of circulating TNNI3K level using the monoclonal-antibodies may be a novel and useful tool to diagnose AMI. Furthermore, both ELISA and gold colloid methods are used to observe their sensitivity and specificity. Results showed that the sensitivity and specificity of anti-TNNI3K monoclonal antibody were higher than other biomarkers anti-TNNI3K monoclonal antibody and the measurment time in the gold colloid method is significantly shortened when compared with using ELISA method. In conclusion, using TNNI3K as a molecular target is a new potential approach for developing therapeutic or diagnostic agents for ischemic cardiac diseases by means of molecular biological techniques.
Abstract 271: Id3 Promotes Obesity-Induced Inflammatory Macrophage Accumulation Through Proliferation of MCP-1-Producing Adipocyte Progenitor Cells
Authors: Kaplan Jennifer L , Marshall Melissa A , McSkimming Chantel , Harmon Daniel B , Garmey James , Oldham Stephanie , Hallowell Peter , McNamara Coleen A
Published: March 2022

High fat diet (HFD)-induced increases in MCP-1 are a hallmark feature of adipose tissue inflammation during obesity. Macrophages are the main producers of MCP-1 in established obesity; however, early increases in obesity-induced production of MCP-1 precede migration of M1 macrophages to adipose tissue. Global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates HFD-induced obesity and adipose tissue inflammation. Specifically, Id3-/- mice have less visceral adipose tissue (VAT), fewer M1 adipose tissue macrophages, and attenuated HFD-induced MCP-1 production. The source of initial obesity-induced MCP-1 in vivo, as well as the role that Id3 plays in regulation of MCP-1, is currently unknown.

Flow cytometry demonstrated that committed CD45-CD31-Ter119-CD29+CD34+Sca-1+CD24- adipocyte progenitor cells (AdPCs) express high levels of MCP-1. HFD increased the number of AdPCs in an Id3-dependent manner. Loss of Id3 increased levels of p21Cip1 and attenuated HFD-induced proliferation of AdPCs, resulting in lower levels of MCP-1 and reduced M1 macrophage numbers in VAT, compared to Id3+/+ littermate controls. Adoptive transfer of 50,000 Id3+/+ AdPCs into Id3-/- recipient mice restored adipose tissue inflammation via increased MCP-1 levels and M1 macrophage accumulation in VAT. Notably, the M1:M2 macrophage ratio significantly increased due to injection of AdPCs. Additionally, flow cytometry identified MCP-1-producing CD45-CD31-CD34+CD44+CD90+ AdPCs in human omental and subcutaneous adipose tissue. The percentage of AdPCs expressing MCP-1 was higher in omental adipose compared to subcutaneous adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in omental adipose tissue.

The present study provides the first in vivo evidence that committed AdPCs are the source of early obesity-induced MCP-1. Id3 is a critical regulator of p21Cip1 expression and proliferation in AdPCs, and in this manner, promotes overall MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.
Abstract 278: Vascular Anatomic and Physiologic Relationships with Local Adipose Phenotype in Chronic Kidney Disease Patients
Authors: Sharma Gaurav , Tao Ming , Ding Kui , Longchamp Alban , Hamburg Naomi , Palmisano Joseph N , Dember Laura M , Berceli Scott A , Ozaki C. Keith , Vita Joseph A ,   Hemodialysis Fistula Maturation Study
Published: March 2022

Introduction:
Local adipose biomarkers have recently been shown to correlate with outward remodeling (adiponectin, IL-8, MCP-1, resistin and TNF) and flow volume (adiponectin, MCP-1) after human hemodialysis AVF creation. As a potential mechanism underlying this observation, we hypothesized that vascular anatomy and physiologic function is dependent on local adipose phenotype.

Methods:
During AVF creation surgery, adipose tissue contiguous to the proximal anastomosis was obtained from patients at two institutions participating in the NIDDK Hemodialysis Fistula Maturation (HFM) Study (n=112). Nine adipose-associated biologic mediators (adiponectin, IL-1 beta, IL-6, IL-8, leptin, MCP-1, PAI-1, resistin, and TNF) were quantified. Levels of adipose biomarkers were correlated to measurements obtained from comprehensive pre-operative non-invasive vascular anatomic and functional studies performed as part of the HFM Study protocol using Spearman correlation (before logarithmic transformation) and Pearson correlation (after transformation). Bonferroni correction was performed for multiple testing (alpha/9=0.006)

Results:
Smaller brachial arterial diameter correlated with higher IL-8 (r=-0.23, 95% CI -0.41 to -0.03, p=0.02), MCP-1 (r=-0.24, 95% CI -0.42 to -0.05, p=0.01), and PAI-1 (r=-0.25, 95% CI -0.42 to -0.05, p=0.01) levels and lower brachial arterial flow velocity correlated with higher MCP-1 (r=-0.22, 95% CI -0.40 to -0.02, p =0.03), but the associations were not significant after Bonferroni adjustment. Brachial artery flow- and nitroglycerin-mediated dilation (FMD and NMD), carotid-radial and -femoral pulse wave velocity (PWV), augmentation index, and the slope of the venous volume-pressure relationship did not correlate with adipose mediator levels.

Conclusions:
While there was suggestion of a relationship between local adipose mediator levels and baseline brachial artery characteristics, relationships with vascular function as assessed by FMD, NMD, PWV and venous capacitance slope were not evident using tests of monotonic associations. Processes not reflected in these measures of vascular characteristics and function may underlie relationships between adipose phenotype and vascular remodeling in human AVFs.
Abstract 283: p27
kip1
Dose Affects Collateralization Through Arterial Wall Cells
Authors: Eliahoo Galit A , Bates Olivia , Cox Timothy C , Tang Gale L
Published: March 2022

Objectives:
p27 deficiency (p27
-/-
) improves blood flow after hindlimb ischemia through enlargement of a bridge collateral pathway. p27
-/-
VSMC both migrate and proliferate more than wt VSMC. We hypothesized that p27’s effect on ischemic collateralization is from arterial wall cells and not from bone marrow derived cells.

Methods:
p27
-/-
and wt (C57BL/6) after reciprocal bone marrow transplantation (BMT) and p27
+/-
female mice underwent left femoral artery ligation. Footpad laser Doppler perfusion imaging was performed weekly. MicroCT scans were performed on both hindlimbs after sacrifice (days 14 and 28 for BMT and p27
+/-
mice respectively). VSMC were isolated from all groups and used in cell proliferation, migration, and gel contraction assays.

Results:
p27
-/-
mice transplanted with wt bone marrow recovered significantly more blood flow and enlarged bridge collaterals than wt mice transplanted with p27
-/-
bone marrow (117±15 vs. 55±7 μm, p<0.001). p27
+/-
mice had intermediate blood flow recovery and had intermediate bridge collateral size (wt 82 ± 22, p27
+/-
122±15, and p27
-/-
158±18 μm,
p
< 0.05, Figure 1).
In vitro
studies showed that p27
+/-
VSMC proliferated more but did not migrate more than wt VSMC.

Conclusions:
p27 deficiency enhances ischemic collateralization via effects on local arterial wall cells rather than on bone marrow derived cells. This may be secondary to increased VSMC proliferation.

Figure 1. MicroCT scans of the ischemic hindlimb of p27
-/-
, p27
+/-
and wt mice showing enlargement of bridge collaterals in p27
-/-
more than p27
+/-
mice, and absence of this pathway in wt mice. Digital pseudocoloring has been added (magenta-femoral, saphenous, popliteal arteries, blue-collateral arteries, red-unnamed muscular arteries). L-ligation site, 1-bridge collaterals, 2-gracilis collaterals.
Abstract 285: Rapid Change In Red Blood Cell PUFA Composition With High-dose Fish Oil Supplementation In Patients With Peripheral Artery Disease (PAD).
Authors: Grenon Marlene , Alley Hugh , Hall Christine , Perez Sandra , Gasper Warren , Harris William , Conte Michael S , Owens Christopher
Published: March 2022

Introduction:
Current guidelines recommend a diet rich in fish or n-3 polyunsaturated fatty acid (PUFA) supplementation in patients with cardiovascular disease including PAD. The omega-3 index represents the red blood cell (RBC) content of the two major long-chain n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with less within-patient variation compared to plasma levels of n-3 PUFA. We aimed to quantify the change in the omega-3 index with high-dose n-3 PUFA supplementation in a PAD cohort.

Methods:
This study was a secondary analysis of the OMEGA-PAD I Trial (NCT01310270), a randomized, double-blinded, placebo-controlled trial addressing the hypothesis that short-duration, high-dose n-3 PUFA oral supplementation improves endothelial function and inflammation in subjects with PAD. Eighty patients with stable claudication received 4.4g of fish oil (2.6g of EPA + 1.8g of DHA) daily (n=40) or placebo capsules (n=40) for 1 month. The RBC fatty acids (FA) content was measured by gas chromatography (OmegaQuant Analytics, Sioux Falls, SD) and expressed as a percent of total FA.

Results:
There was no difference in baseline PUFA content between the two groups. There was a shift in the n-6:n-3 ratio in RBC from 5 ± 1 to 3 ± 1 (p<0.0001) and the omega-3 index increased from 5 ± 1 to 9 ± 2 (p<0.0001) in the fish but not in the placebo group (Table). There was a significant increase in EPA, DHA, and n-3 docosapentaenoic acid and a significant decrease in all n-6 PUFA content of RBC in the fish oil but not in the placebo group. Following treatment, there was also a significant reduction in plasma triglycerides (-34 ± 46, p<0.001) in the fish oil group. The change in triglycerides was significantly correlated with the change in omega-3 index (p=0.03).

Conclusions:
High-dose fish oil supplementation increases the omega-3 index and reduces n-6 PUFA levels. The implications of these changes for patients with PAD will continue to be investigated in a follow-up trial (OMEGA-PAD II).
Abstract 300: A Biodegradable Wrap for Peri-vascular Delivery of Pro-resolving Lipid Mediators
Authors: Wu Bian , Lance Kevin D , Chatterjee Anuran , Mottola Giorgio , Chen Mian , Komshian Sevan R , Desai Tejal A , Conte Michael S
Published: March 2022

Introduction:
Persistent inflammation following vascular injury leads to excessive scarring, limiting the success of vascular interventions. Recent work has identified that endogenous specialized proresolving lipid mediators (SPM) such as resolvin D1 (RvD1) actively orchestrate the process of resolution, exerting vasculo-protective effects without associated toxicity. We propose local vascular delivery of SPM through a biodegradable wrap.

Methods:
RvD1 (200 ng) was heat-sealed between thin layers of polycaprolactone (PCL) or poly-lactic-co-glycolic acid (PLGA). PLGA membranes differed in their composition of lactic versus glycolic acid (%-lactide). Directional drug release was measured via EIA in a cell-free system in vitro and into rabbit aortas exposed to pulsatile flow ex vivo. Bioactivity was confirmed on human vascular smooth muscle cells (VSMC) using migration and proliferation assays.

Results:
Of the constructs tested, a 3-layered PLGA wrap consisting of 85%/75%/50%-lactide provided the most favorable drug elution (Fig1a), with sustained release of >800 pg/day for at least 14 days and nearly all elution occurring from the 50%-lactide side (Fig1b). Perivascular application of this wrap ex vivo with the 50%-lactide side facing “in” demonstrated uptake into rabbit aortic walls at 8 hours (0.4 ± 0.1 pg/mg). VSMC cultures exposed to drug-loaded wraps showed inhibition of migration (40%) and proliferation (25%).

Conclusion:
We demonstrate sustained and directional elution of therapeutically relevant amounts of biologically-active RvD1 through a biodegradable perivascular wrap, providing opportunity for translational studies of SPM in vascular injury.
Abstract 301: Resistin is an Independent Indicator of Inflammation in Patients Undergoing Carotid Interventions
Authors: Zuniga Mary C , Hitchner Elizabeth , Raghuraman Gayatri , Zhou Wei
Published: March 2022

Objective:
Resistin has been associated with increased risk for cardiovascular complications. However, the underlying mechanisms are not yet fully elucidated. This study aimed to investigate whether resistin modulated plasma cytokines, chemokines, and growth factors in patients undergoing carotid revascularization procedures.

Methods:
Patients undergoing carotid artery stenting (CAS) and carotid endarterectomy (CEA) at a single institution were recruited to participate. Blood plasma collection was performed within 48 hours following intervention using an established protocol, and demographics were recorded. Samples were stored at -80 °C and analyzed with a Luminex magnetic beads-based assay. Patients were divided into tertiles based on plasma resistin concentration (mean pg/mL): low (2237.6, n=30), medium (4252.3, n=31), and high (7467.5, n=30). Levels of cytokines were compared using ANOVA.

Results:
91 male patients with a mean age of 69 years were included (50 CAS, 41 CEA). Most patients had a history of smoking (84%). Medical comorbidities included hypertension (93%), diabetes (45%), CAD (49%), and obesity (44%). The distribution of comorbidities was similar within each tertile. The levels of resistin were not significantly different in CAS vs CEA patients. There was also no significant difference in various growth factors (VEGF-A, M-CSF, and GM-CSF), neurotrophic factors (BDNF and β-NGF), or adhesion molecules (ICAM-1 and VCAM-1). However, significantly higher levels of the pro-inflammatory cytokines/chemokines were observed in the high resistin tertile compared to the low tertile: TNF-α (p < 0.001), IL-6 (p < 0.05), IL-12p40 (p < 0.01), IL-12p70 (p < 0.05), MIP-1α (p < 0.05), MIP-1β (p < 0.05), and MCP-3 (p < 0.01). Similarly, significantly higher levels of the aforementioned pro-inflammatory cytokines (except MCP-3) were observed in the high compared to the medium tertile.

Conclusions:
Patients with higher resistin levels exhibit higher levels of inflammatory profiles post-intervention independent of procedure type or comorbidities, implicating that resistin-induced inflammation may be a mechanism for resistin-related vascular complications.