bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 13: Macrophage Erk5 Activation Induced by Apoptotic Cells and Statins Promotes Efferocytosis and Inhibits Atherosclerosis Formation
Authors: Heo Kyungsun , Cushman Hannah , Woo Chang-Hoon , Akaike Masashi , Wang Xin , Morrell Craig N , Fujiwara Keigi , Abe Jun-ichi
Published: July 2021

Backgrounds

Defective efferocytosis (phagocytic clearance of dying cells) is critically linked to progression of advanced atherosclerotic lesions. Although it has been well known that efferocytosis is processed by molecules including eat-me and find-me signals, it is unclear how efferocytosis becomes defective in advanced lesion.

Methods and Results

Here, we found that bone marrow-derived macrophages (BMDM) from macrophage-specific ERK5 knockout (ERK5

fl/fl

LysM
Cre
+/-
; ERK5-MKO) mice reduced mRNA and proteins levels of efferocytosis-related signaling molecules such as Mer-tK, C1qa, C1qb, C1qc, Gas6, Mfg-e8, Thbs1, and Anxa1 compared with BMDM from non-transgenic control (NLC) mice. Interestingly, addition of apoptotic cells and pitavastatin activate ERK5 kinase activity and increase opsonins, eat-me and find-me signals, and phagocytic capacity toward apoptotic cells in normal macrophage, but macrophages from ERK5-MKO failed to respond in this manner. ERK5-MKO crossed to LDLR
-/-
mice and fed a high cholesterol diet for 16-week accelerated atherosclerosis formation with an increased level of apoptotic cell accumulation and necrotic core formation, which was accompanied by a significant reduction in collagen content and fibrous cap thickness. We also found lower levels of expression for Mfg-e8 and Thbs1 in the advanced necrotic core of ERK5-MKO mice compared to NLC mice.

Conclusion

Our study shows that apoptotic cells and statins-activated ERK5 plays a key role in coordinating the process of efferocytosis. Our results provide a mechanistic understanding of the clinically well-described cardiac risk of acute atherothrombotic events in advanced lesions.

1
Abstract 144: A20 Inhibits Pathologic Interferon-? Signaling in Smooth Muscle Cells to Prevent Atherosclerotic Vascular Disease
Authors: Moll Herwig P , Lee Andy , Ferran Christiane
Published: July 2021

The pleiotropic cytokine Interferon-γ (IFNγ), best known for its anti-viral and immunomodulatory activities, also demonstrates pro-atherogenic effects. Implication of IFNγ in the pathogenesis of atherosclerosis results in part from the upregulation of a number of pro-inflammatory interferon stimulated genes (ISGs) in vascular smooth muscle cells (SMC). Hence, strategies to reduce IFNγ signaling are of particular interest to reduce or prevent pathologic vascular remodeling associated with atherosclerotic disease, for instance transplant arteriosclerosis.

We discovered that the anti-inflammatory and vasculoprotective protein A20 inhibits IFNγ signaling and thus the expression of ISG in vitro in human coronary artery SMC, and in vivo in vascular allografts. The aim of this study was to unveil the mechanism(s) of A20 mediated inhibition of IFNγ signaling.

Adenovirus-mediated overexpression of A20 in SMC significantly decreased mRNA and protein levels of signal transducer and activator of transcription 1 (STAT1), the key mediator of IFNγ signaling, as a result of lower transcription. Moreover, overexpression of A20 in SMC also efficiently inhibited activating Y701 phosphorylation of STAT1, through a mechanism possibly implicating A20 binding to STAT1, as confirmed by co-immunoprecipitation. As expected, overexpression of A20 also inhibited NF-κB activation in SMC, which is required for full-blown IFNγ responses. Indeed, we confirmed that inhibition of NF-κB by overexpression of its inhibitor, IκBα also quenched upregulation of ISGs in SMC, albeit without down-regulating STAT1.

Overall, our results identify A20 as a potent inhibitor of IFNγ signaling in SMC through several non-mutually exclusive mechanisms. This novel function of A20 in SMC further qualifies its atheroprotective properties and supports the pursuit of A20-based therapies for the cure of atherosclerotic vascular disease.
Abstract 152: Stat4 Deficiency limits the Development and Progression of Atherosclerosis
Authors: Taghavie-Moghadam Paresa , Butcher Matthew , Kaplan Mark , Nadler Jerry , Galkina Elena
Published: July 2021

T helper 1 (Th1) cells constitute the majority of plaque infiltrating IFNγ+ T cells and play a pro-atherogenic role. Th1 cells are induced via IFNγ-dependent activation of T-box expressed in T cells (Tbet) and/or IL-12-dependent activation of signal transducer and activator of transcription 4 (Stat4). While the role of Tbet in atherosclerosis is established, the impact of the IL-12/Stat4-dependent pathway is not well defined. To address the role of Stat4 in atherosclerosis, we bred Stat4-deficient mice with Apolipoprotein E-deficient mice to generate Stat4-/-Apoe-/- mice. Deficiency of Stat4 resulted in approximately a 70% reduction in the plaque burden for 34 week old Stat4-/-Apoe-/- mice fed a chow diet and in 12 week old Stat4-/-Apoe-/- mice fed a western diet there was approximately a 40% reduction in plaque burden, both compared with diet matched Apoe-/- controls females (p<0.001). To assess the effect of Stat4 on Th1 and Treg cell differentiation, we performed an in vitro polarization assay. Deficiency of Stat4 reduced differentiation of IFNγ+ Th1 cells in Th1 conditions, but supported the induction of Tregs in Treg polarizing conditions, confirming the importance of Stat4 in regulating the Th1/Treg balance. In contrast to the in vitro results, we found no difference in the expression of both IFNγ and Foxp3 amongst Stat4-/-Apoe-/- and Apoe-/- lymph nodes and splenic CD4+ T cells; suggesting that additional cytokines in vivo may induce IFNγ+Th1 and inhibit Treg differentiation. Stat4 deficiency also resulted in increased splenic B cell numbers and a slight increase in B1a dependent T15/E06 mRNA expression. Stat4 is a powerful regulator of chemokine expression within peripheral tissues. Adoptively transferred Apoe-/- B cells and CD11b+ cells migrated more efficiently into Stat4-/-Apoe-/- aortas compared to Apoe-/- recipients. However, percentages of macrophages, as determined by CD11b+CD68+ were reduced within the spleens and aortas of Stat4-/-Apoe-/- mice as compared to Apoe-/- controls at steady state conditions. In conclusion, Stat4 deficiency results in reduced atherosclerosis via the modulation of B cell function and aortic leukocyte content.
Abstract 157: Association of Cholesterol Profile to the Number of Vessels Involved with Coronary Artery Disease
Authors: Misra Neeladri , Kim Woo Sung , Scavina Michael , Ross Jill
Published: July 2021

Introduction

The cholesterol profile is an important modifiable risk factor for CAD and our understanding of atherosclerosis has helped develop drugs that have significantly impacted treatment outcomes of CAD by lowering cholesterol levels. A study to find a correlation between the cholesterol profile and the number of vessels involved with CAD was our objective.

Hypothesis

Patients with significant CAD (defined as >50% stenosis on coronary angiography) in 2 or more vessels had worse cholesterol profiles compared to single vessel. A subset of the study looked to see if the use of statins affected the severity of CAD.

Methods

A retrospective review of 498 patients from our hospitals cardiac registry was done. The cholesterol profile of these patients done either during the admission for cardiac catherization or the most recent one was used to correlate. The catheterization reports were reviewed to find the number of vessels involved with >50% stenosis and a CAD severity score was calculated using the gensini scoring system. We also reviewed the patients medication reconciliation forms to see if they had been on statins.

Results

We could not correlate between the number of vessels affected with significant CAD and the cholesterol profile. Using One way ANOVA analysis neither the Tc/HDL ratio ( p value 0.692) or LDL (p value=0.458) or the HDL levels( p value=0.588) had any association to the number of coronary vessels affected with CAD. In our study subset, the use of statins did not lower the gensini scores significantly as compared to patients who were not on statins (p value 0.95).

Conclusions

The cholesterol profile bears no correlation with the number of vessels affected by CAD. Based on our findings, taking statins did not significantly influence the severity of CAD.
Abstract 18: The Effect of Lipid Modification on Peripheral Arterial Disease after Endovascular Intervention Trial (ELIMIT)
Authors: Brunner Gerd , Yang Eric Y , Kumar Anirudh , Wensheng Sun , Salim Salim S , Negi Smita , Murray Tyler , Lin Peter H , Hoogeveen Ron C , Chen Changyi , Kougias Panagiotis , Taylor Addison , Lumsden Alan B , Nambi Vijay , Ballantyne Christie M , Morrisett Joel D
Published: July 2021

BACKGROUND

The Effect of Lipid Modification on Peripheral Arterial Disease after Endovascular Intervention Trial (ELIMIT), a prospective double-blind randomized study, was designed to determine over 24-months, the effects of triple drug lipid modification therapy versus mono therapy on the progression of atherosclerotic lesions in the distal superficial femoral artery (SFA), as assessed by 3.0T magnetic resonance imaging (MRI).

METHODS

A total of 95 patients were randomized to either mono therapy with simvastatin (40mg) or triple therapy with simvastatin (40mg), extended-release niacin (1.5g), and ezetimibe (10mg). MR imaging was performed at baseline, 6-, 12-, and 24-months. SFA wall, lumen, and total vessel volumes were quantified using inter-visit co-registered proton-density-weighted turbo spin echo MRI sequences. MRI derived SFA parameters and lipids were analyzed with multi-level models and non-parametric tests, respectively.

RESULTS

Baseline characteristics did not differ between mono and triple therapy groups, except for ethnicity (Table). There was no difference in adverse cardiovascular events between groups (p=0.99). SFA wall, lumen, and vessel volumes increased non-significantly for both groups between baseline and 24-months. Non-high-density lipoprotein cholesterol showed a larger reduction in triple therapy compared with mono therapy at 12-months (p=0.01).

CONCLUSION

Despite improved atherogenic lipid profiles, there was no significant difference between mono therapy with simvastatin and triple therapy with simvastatin, extended-release niacin, and ezetimibe for the 24-month changes in total SFA wall, lumen, and vessel volumes.
Abstract 191: NF-?B p65 Methylation by Protein Arginine Methyltransferase 5 (PRMT5) is Necessary for the Transcriptional Induction of CXCL10 by TNF-a
Authors: Harris Daniel P
Published: July 2021

TNF-α initiates the expression of genes involved in the recruitment, adhesion, and transmigration of leukocytes to sites of inflammation. Here, we report that the protein arginine methyltransferase PRMT5 is required for the transcriptional induction of the pro-inflammatory chemokine CXCL10 (IP-10) in endothelial cells. Depletion of PRMT5 by siRNA results in significantly diminished TNF-α-induced CXCL10 mRNA expression, but does not affect expression of other chemokines, such as MCP-1 or IL-8. Chromatin immunoprecipitation experiments of the CXCL10 proximal promoter show the presence of symmetrical dimethylated arginine (sDMA)-containing proteins upon exposure to TNF-α. This methylation is completely lost when PRMT5 is removed from cells by siRNA. Using immunoprecipitation, we show that PRMT5 enhances CXCL10 expression by methylating the RelA (p65) subunit of NF-κB. In summary, we have identified that PRMT5 is a novel regulator of CXCL10 expression. Further, we have discovered that PRMT5 methylates NF-κB, a finding which may further knowledge of the post-translational code governing NF-κB regulation and target specificity.
Abstract 202: Enrichment of Transcription Factor Binding Sites in the Promoter Regions of Differentially Expressed Genes in Vascular Smooth Muscle Cells from Abdominal Aortic Aneuryms.
Authors: Airhart Nathan , Curci John
Published: July 2021

Background

We have previously shown that VSMC from AAA are unique compared to cells from normal aorta (NAA) and carotid endarterectomy (CEA) with increased production of matrix metalloproteinases and elastin degrading activity. The purpose of this study was to explore the mechanisms behind this phenotype.

Methods

Tissue for VSMC cultures was obtained from patients undergoing AAA repair and CEA. NAA tissue was obtained from renal transplant patients (NAA). Total RNA was isolated from VSMC and subjected to whole-genome microarray. Enrichment of binding sites for transcription factors (TF) within 5 kD of transcription start sites of upregulated genes were identified using Whole Genome rVista. Enriched gene ontology terms were identified using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).

Results

Gene profiles of 22 AAA, 29 CEA, and 17 NAA cell lines were compared. We identified 120 upregulated genes in AAA-VSMC relative to NAA and CEA-VSMC (FDR<0.05). Analysis of transcription factor binding sites of these genes showed enrichment of TFs including members of the ETS, AP-1, and Rel/Ankyrin families. Gene ontology (GO) revealed enrichment of developmental process and immune system genes. Analysis by cell compartment showed enrichment of extracellular matrix and intermediate filament cytoskeleton genes (Table 1).

Conclusion

This is the first study to demonstrates enrichment of TF families such as ETS, AP-1 and Rel-Ankyrin in AAA VSMC. This suggests that VSMC in AAA may not just be responding to inflammatory or other local stimuli, but may be directly contributing to the ECM changes that define AAA.
Abstract 208: Altered Cerebrovascular Reactivity Following One Month of Obstructive Sleep Apnea
Authors: Durgan David J , Crossland Randy F , Lloyd Eric E , Phillips Sharon C , Bryan Robert
Published: July 2021

Obstructive sleep apnea (OSA) has been identified as a significant risk factor for stroke. However, little is known regarding the effects of OSA on the cerebrovascular wall. Using a novel rodent model of OSA we assessed the hypothesis that (1) OSA augments endothelin (ET-1) induced constrictions of cerebral arteries and (2) OSA attenuates dilations of cerebral arteries by agonist-induced nitric oxide (NO) release from the cerebrovascular endothelium. The repetitive airway closures associated with OSA lead to intermittent hypoxia/hypercapnia and reoxygenation, increased negative intrathoracic pressures, and arousals. In order to model the physiological consequences of OSA, we have chronically instrumented rats with inflatable endotracheal obstruction devices. Unanesthetized freely-ranging rats underwent 30 apneas/ hour for 8 hours/ day (sleep phase) for 1 month. During apnea pO
2
decreased from 122±3 to 67±3 mm Hg; pCO
2
increased from 43±1 to 51±1 mm Hg; pH decreased from 7.46±0.00 to 7.38±0.01; and hemoglobin O
2
saturation decreased from 94±1 to 82±1 % (n=5 and p<0.05 for each). Following 1 month of OSA blood pressure, plasma ET-1 and NO levels were similar in sham and OSA rats. Using the pressurized cerebral artery preparation, we observed a 17.5-fold increase in sensitivity to ET-1 (n=5-6, p<0.05) after 1 month of OSA. The increased sensitivity of OSA cerebral arteries to ET-1 was abolished by the ET-B receptor antagonist BQ-788 (n=6, NS). Additionally, constrictions to the ET-B specific agonist IRL-1620 were significantly greater in OSA, versus sham, cerebral arteries (n=6, p<0.05). Dilations to ATP (a P2Y
2
agonist which stimulates NO production in the endothelium) were attenuated in cerebral arteries from OSA rats by 40% (n=5-8, p<0.05). However dilations to the NO-donor MAHMA-NOnoate were similar between groups. In conclusion, 1 month of OSA results in (1) increased sensitivity of cerebral arteries to ET-1, likely through upregulation of ET-B receptors on the vascular smooth muscle and (2) decreased endothelial-derived NO production. These data suggest that OSA results in significant alterations to the cerebrovascular wall in the absence of hypertension.
Abstract 230: Effect of aerobic exercise on Endothelial Progenitor Cells (EPCs) in patients with prediabetes
Authors: Sen Sabyasachi , Chou Cyril , Pollard Nicole , Islam Ashequl
Published: July 2021

Introduction

At least 79 million Americans have pre-diabetes. Though life-style modification prevents progression from Pre-diabetes to overt diabetes, the effect on vascular endothelial progenitor cells (EPC) stem cells is unknown.

Methods

This is a crossover study of 16-week duration, using exercise-naive pre-diabetes patients, n=12, aged 45-65 yrs with a BMI of 25-34.9. We studied flow mediated dilatation (FMD), EPC function and gene expression and serum endothelial inflammatory levels after 6 weeks each of aerobic exercise (150min/week) and non exercise phase, in a cross over design with 4 week wash-out period between the 2 phases. EPC migration was noted in response to chemotactic factors such as Vascular Endothelial Growth Factor (VEGF-A, 0- 50ng/ml) and Stromal Cell-Derived Factor-1(SDF1α, 0-100 ng/ml). Exercise compliance was monitored by regular phone calls and downloadable accelerometers.

Results

FMD studies (undertaken by 3 observers) showed mean FMD in non-exercise group of 5.7+0.6% which improved to 11.2+0.9% post-exercise. No significant weight loss noted between the 2 groups, however, significant reduction in leptin, IL-6, TNFα, hs-CRP, Triglyceride, and ApoB levels was noted. EPC gene expression analysis showed decrease of eNOS (5 fold) and increase of PECAM-1 (4-fold), Endothelin-1 (3-fold), IL-6 (2-fold), TNFα (3-fold) in non-exercise which improved following exercise. EPC migration improved post exercise, particularly in response to SDF-1α as did CD34+ cell number and EPC colony formation improved significantly also.

Conclusion

We demonstrate that pre-diabetic state is associated with poor vascular reactivity and impaired EPC stem cell function. Pre-diabetes may be a clinical window of therapeutic intervention opportunity when short course of aerobic exercise may allow improvement of vascular reactivity and EPC function despite no statistically significant weight loss. This study also indicates EPCs may act as an important biomarker for vascular function.
Abstract 231: Bone Marrow-Derived Mesenchymal Stem Cells from Amputated Limbs of Patients with Critical Limb Ischemia Provide Stromal Support for Endothelial Cells
Authors: Wang Ruoya , Copland Ian , Li Haiyan , Peister Alexandra , McDevitt Todd , Brewster Luke
Published: July 2021

Critical limb ischemia (CLI) often results in amputation, but autologous cellular therapy using bone marrow-derived mesenchymal stem cells (MSCs) may be able to prevent this outcome. However, the quality of MSCs in the CLI population is not clear. Amputated CLI limbs contain reservoirs of MSCs that can be used to characterize their stromal capacity. We hypothesize that MSC stromal capacity is relatively preserved in ischemic limbs compared to ones from healthy donors.

Methods

Healthy donor MSCs (hdMSCs, n=2) and ischemic MSCs (iMSCs, n=3) from amputated limbs of non-diabetic CLI patients were cultured. Human microdermal ECs were commercially obtained. Co-culture pellets (1:1 ratio) of hdMSC:EC, iMSC:EC, and EC alone were formed and cultured in 3-D fibrin hydrogels in EC growth media. Images of the pellets and invasion areas were captured up to 5 days. The invasion area was quantified as the difference between the total and pellet area normalized to initial pellet area. Experiments were performed in quadruplicates and co-culture experiments were duplicated.

Results

iMSC:EC co-culture provided similar stromal support as the healthy donor cohort. Both MSC groups were significantly greater than the ECs alone following day 2 (see figure, *P<0.0005, ANOVA). Both MSC groups also exhibited significant increase in invasion area with time (P<0.01) while the ECs did not (P>0.5).

Conclusion

This is the first study to demonstrate that iMSCs provide similar stromal support to ECs as healthy hdMSCs, thus iMSCs may be of sufficient quality for use in autologous therapy. We are currently quantifying secretome expressions of the iMSCs to provide insights into mechanisms of the stromal support.
Abstract 27: Dok-1 Negatively Regulates Integrin aIIbß3 Outside-in Signaling and Inhibits Thrombosis
Authors: Niki Masaru , Jin Hong , Chauhan Anil K , Lentz Steven R
Published: July 2021

Background

Platelet agonists activate integrin αIIbβ3 to allow the binding of soluble fibrinogen (a process known as inside-out signaling). Subsequent platelet aggregation leads to αIIbβ3 outside-in signaling, which results in tyrosine phosphorylation of β3 and other proteins, cytoskeletal reorganization, and platelet spreading. It has been reported that the adapter protein Dok-1 binds to the cytoplasmic tail of β3 and inhibits αIIbβ3 activation, but the specific role of Dok-1 in regulating inside-out or outside-in platelet signaling remains undefined.

Methods

We assessed the effects of Dok-1 on platelet signaling and thrombosis in Dok-1 null (Dok-1-/-) mice. Inside-out signaling was assessed by measuring αIIbβ3 activation (using the JON/A antibody) and fibrinogen binding by flow cytometry after stimulation of platelets with thrombin, ADP, and/or the thromboxane A2 receptor agonist
U46619.
Outside-in signaling was examined by measuring platelet spreading and clot retraction. Tail-transection bleeding time and susceptibility to thrombotic occlusion of the carotid artery in response to photochemical injury (rose bengal) were also measured.

Results

No significant differences in JON/A or fibrinogen binding were detected between wild-type (Dok-1+/+) and Dok-1-/- platelets, suggesting that Dok-1 does not regulate inside-out signaling. In contrast, Dok-1-/- platelets exhibited increased clot retraction and enhanced spreading on fibrinogen upon thrombin stimulation compared to Dok-1+/+ platelets (P<0.05), suggesting that Dok-1 negatively regulates outside-in signaling. Compared with Dok-1+/+ mice, Dok-1-/- mice had shorter bleeding times (181±168 vs. 379±193 seconds; P<0.001) and Dok-1-/- mice had shorter times to stable occlusion times of the carotid artery after photochemical injury compared with Dok-1+/+ mice (16.4±6.1 vs. 25.0±8.1 minutes; P<0.05).

Conclusions

The adaptor protein Dok-1 functions to negatively regulate integrin αIIbβ3 outside-in signaling. Deficiency of Dok-1 results in a prothrombotic phenotype, with shorten bleeding times and enhanced arterial thrombosis.
Abstract 280: Inhaled Carbon Monoxide Attenuates Aortic Aneurysmal Growth in a Topical Elastase Mouse Model
Authors: Shukla Ankur J , Washington Chris , McEnaney Ryan , Tzeng Edith
Published: July 2021

Objective

Abdominal aortic aneurysms (AAA) affect up to 9% of men over 65 years of age with increasing incidence with further aging. Little is still known about the pathogenesis of AAA but inflammation appears to be one of the main contributing factors. Carbon monoxide (CO) is an endogenously produced molecule that has been shown to have significant anti-inflammatory and cytoprotective properties. Thus, we hypothesize that exogenous CO may attenuate AAA growth.

Methods

Eight week-old C57Bl6 mice were anesthetized and infrarenal aorta were exposed, measured in 3 locations, and treated with topical porcine pancreatic elastase (30 ul). Mice were either maintained in room air for the duration of the experiment or treated with inhaled CO (250 PPM) for 1 hr daily beginning on day 1. Animals were sacrificed on day 14 when aortic diameters were measured and tissues collected for morphometric analysis.

Results

Topical elastase treatment resulted in marked aortic enlargement with true aneurysm formation (253% of baseline, N=8). However, CO treatment reduced aneurysm size significantly (222%, N=8; p-0.039) with noticeable reduction in adhesion formation and scarring around the aorta. Histologically, CO treated animals maintained a larger medial area than controls (0.092 mm
2
vs 0.057 mm
2
; p = 0.023) (Figure 1).

Conclusion

Inhaled CO appears to mitigate aneurysmal degeneration of the abdominal aorta induced by topical elastase. There was a clear reduction in adhesions to the aorta, strongly supporting the anti-inflammatory mechanism involved. The maintenance of a thicker medial component of the aorta in CO treated mice also suggests protection of the smooth muscle cells.
Abstract 301: Angiotensin II-induced Aortic Stiffening is Associated With Upregulation of Mir-21 And Downregulation of Mir-29b
Authors: Raaz Uwe , Toh Ryuji , Adam Matti , Nakagami Futoshi , Maegdefessel Lars , Deng Alicia , Spin Joshua M , Tsao Philip S
Published: July 2021

Background

Arterial stiffening is associated with aging, as well as numerous diseases such as hypertension, congestive heart failure, diabetes mellitus and renal failure, and has been identified as an independent risk factor for cardiovascular disease. Consequently there is a growing interest in the mechanisms that govern the process of arterial remodeling. Recently, small noncoding RNAs, termed microRNAs (miRs), have emerged as powerful cellular regulators involved in disease and tissue remodeling. More specifically, miR-29b downregulation as well as miR-21 upregulation have been identified as pro-fibrotic mechanisms in various cardiovascular disease models. This study investigated the role of miR-21 and miR-29b in a mouse model of AngII-induced arterial remodeling.

Materials and methods

Angiotensin II (AngII) (1000ng/kg/min) was infused via osmotic pumps in 10-week-old apoE-/- male mice (C57BL/6J background) for 7 days. Subsequently, arterial stiffness was determined in vivo using ultrasound-based measurements. Abdominal aortas were harvested and expression of various collagen isoforms (Col1a1, Col3a1, Col5a1) known to be crucial determinants of arterial remodeling/stiffening was quantified via qRT-PCR. We also measured expression levels of miR-21 and miR-29b.

Results

AngII stimulation resulted in an increased aortic stiffness paralleled by a marked pro-fibrotic response as evidenced by significant increases in Col1a1, Col3a1, and Col5a1 in the infrarenal aorta compared to baseline levels (p < 0.05). This increase was accompanied by significant downregulation of miR-29b, and upregulation of miR-21 (p <0.05).

Conclusion

The pro-fibrotic response in AngII-mediated arterial remodeling is associated with an increase in miR-21 and a decrease in miR-29b. Modulation of miR-21 and miR-29b have both been successful in altering fibrotic mechanisms in various cardiovascular diseases. These data suggest they may also be potential targets in the treatment of hypertensive vascular remodeling/arterial stiffening.
Abstract 309: Osteogenic Induction of p-ERK 1/2 Signaling Associated with NOTCH1 Inactivation in Human Aortic Valvar Interstitial Cells
Authors: Godby Richard C , Munjal Charu , Opoka Amy M , Narmoneva Daria A , Hinton Robert B
Published: July 2021

Introduction

Heart valve disease affects over 2% of the population, leading to at least 20,000 deaths and $1 billion in direct costs in the US each year. Calcific aortic valve disease (CAVD) is linked to loss of function mutations in
NOTCH1
that result in osteogenic gene induction.
In vitro
animal studies have established that multiple osteogenic targets are mediated through induction of p-ERK 1/2 signaling; however, the relationship between NOTCH1 and p-ERK has not been examined in primary human aortic valvar interstitial cells (AVICs).

Hypothesis

We tested the hypothesis that chemically-induced NOTCH1 suppression results in mineralization due to p-ERK 1/2 activation in human AVICs.

Methods

Human AVICs were isolated from CAVD specimens at the time of valve replacement. AVICs were cultured for two weeks in complete media containing DMEM and 10% FBS, and treated with calcific media (10mM b-glycerol phosphate; 60μM ascorbic acid-2-phosphate; 1μM dexamethasone), which has been shown to suppress NOTCH1 signaling. Standard real-time RT-PCR, western blot, and immunocytochemistry were used to compare groups.

Results

Treatment with calcific media resulted in a 2.3 fold decrease in the level of
NOTCH1
expression and a 3 fold increase in p-ERK 1/2 expression, assessed with both western blots and immunocytochemistry. Interestingly, calcific media also induced a 3.5 fold decrease in VEGF, consistent with early osteogenic differentiation. Additionally, the AVICs treated with calcific media demonstrated overt calcification by Alizarin Red staining.

Conclusions

Human AVICs demonstrated calcification associated with p-ERK 1/2 activation and reduced
NOTCH1
levels, suggesting p-ERK inhibition may mitigate the effects of NOTCH loss of function, thereby representing a potential pharmacologic therapy for CAVD. Further investigation of the molecular mechanisms linking NOTCH1 and p-ERK are warranted, including studies that specifically inhibit NOTCH1 signaling and define the impact of mechanical forces on molecular pathways, thereby identifying mechanisms underlying CAVD progression.
Abstract 311: TGFb/Smad3 Stimulation Drives Smooth Muscle Cell De-differentiation
Authors: DiRenzo Daniel M , Shi Xudong , Seedial Stephen , Guo Lian-wang , Liu Bo , Kent K. Craig
Published: July 2021

Restenosis, or vessel re-narrowing, occurs in approximately 25-50% of arterial interventions involving balloon angioplasty due to the formation of a proliferative plaque in the vessel lumen termed neo-intimal hyperplasia. Arterial smooth muscle cells (SMCs) contribute to neo-intimal hyperplasia through a de-differentiation process that includes downregulation of their contractile gene expression and conversion to a phenotype that includes proliferation, migration, and matrix synthesis. Expression of TGFβ and its downstream signaling protein, Smad3, are greatly upregulated following vascular injury, including balloon angioplasty. Classically, TGFβ signaling has been shown to suppress SMC proliferation and migration in vitro, however, Smad3 overexpressing SMCs demonstrate enhanced proliferation and migration. Furthermore, overexpression of Smad3 in rat carotid arteries enhances neo-intimal hyperplasia following balloon angioplasty. These results lead us to hypothesize that TGFβ signaling, in the context of upregulated Smad3, drives SMC de-differentiation leading to enhanced cellular proliferation and migration.

We utilized primary rat SMCs infected with adenovirus constructs overexpressing Smad3 or GFP control and performed gene expression microarrays 24 hours following TGFβ administration. We observed statistically significant (p<0.05) upregulation of 145 genes and downregulation of 76 genes by more than 3-fold. GO term analysis revealed that genes involved in embryonic tissue development (41 genes) and stem/progenitor cell differentiation (27 genes) were significantly enriched in TGFβ/Smad3 stimulated cells. Confirmatory qRT-PCR demonstrated that the contractile genes SM-MHC, smooth muscle actin, and calponin were significantly downregulated -6.3, -2.7 and -2.1 fold, respectively. In contrast, stem/developmental related genes Cxcr4, Cd34, Wnt11, Wnt2b and IL11 were significantly upregulated by 105.2, 22.3, 11.5, 14.0, and 12.5 fold, respectively. These results strongly suggest that TGFβ/Smad3 stimulation is a powerful de-differentiation signal in SMCs and plays an important role in the development of neo-intimal hyperplasia.
Abstract 320: Heterogeneous Coronary Stent Inflammation and Healing Revealed by in vivo Optical Molecular-Structural Imaging
Authors: Osborn Eric A , Hamidi Ehsan , El Maghraby Amr , Yoo Hongki , Mauskapf Adam , Tearney Guillermo J , Jaffer Farouc A
Published: July 2021

Introduction

Patients with diabetes mellitus that receive coronary stents experience greater stent restenosis and thrombosis, leading to adverse clinical outcomes. Impaired stent healing is linked to elevated endovascular inflammation, but
in vivo
data is lacking. Here, we assessed stent inflammation and tissue healing in diabetic rabbits, using intravascular molecular-structural near-infrared fluorescence (NIRF)-optical frequency domain imaging (OFDI).

Methods

A bare-metal stent (3.5x12mm) was implanted in the infrarenal aorta of alloxan-induced diabetic rabbits (n=5). At day 28, intravascular NIRF-OFDI was performed. Prosense VM110 (5mg/kg IV 24 hrs before imaging; ex/em 750/780 nm) enabled NIRF molecular imaging of inflammatory cathepsin activity. Structural OFDI stent endothelialization (coverage) was simultaneously assessed in 0.5mm intervals. Aortas were then harvested for
ex vivo
fluorescence reflectance imaging (FRI), electrochemical stent dissolution, and cathepsin B immunostaining.

Results

At day 28, stent NIRF inflammatory cathepsin protease activity was enhanced compared to the non-stented aorta (50.9±2.0 vs. 26.1±0.6 nM; p<0.0001). OFDI demonstrated greater average stent edge restenosis (proximal and distal 2mm stent edge neointimal area, 1.63±0.12 vs. mid 2 mm stent 0.50±0.05 mm
2
; p<0.0001), with less de-endothelialized struts at the stent edges (2.1±1.7% vs. 14.0±4.1% mid stent; p=0.03). Stent edge NIRF inflammation was also greater (60.8±3.4 vs. 40.8±1.4 nM mid stent; p=0.0005), and correlated with neointima formation (R=0.43; p=0.003). Conversely, NIRF inflammation negatively tracked with OFDI-uncovered stent struts (R=-0.42; p=0.004). FRI corroborated the enhanced stent-edge NIRF pattern. Matched histopathology revealed cathepsin B expression in NIRF-positive regions.

Conclusions

Intravascular NIRF-OFDI molecular-structural imaging demonstrates that NIRF inflammatory protease activity is linked to increased bare-metal stent edge neointimal formation, but inversely relates to uncovered stent struts. This translatable approach provides new insights into stent inflammation and healing, and may ultimately inform the risk of stent restenosis and thrombosis.
Abstract 323: Increased Levels of Plasma Soluble Sema4D in Patients with Heart Failure
Authors: Lu Qiongyu , Dong Ningzheng , Yi Wenxiu , Wang Yuxin , Zhang Shengjie , Tang Xiaorong , Wu Qingyu , Brass Lawrence , Zhu Li
Published: July 2021

Introduction

Semaphorin 4D (Sema4D/CD100) is a 150-kDa transmembrane glycoprotein expressed by platelets and T-cells. When these cells are activated, Sema4D is cleaved proteolytically, generating a biologically active 120-kDa fragment (soluble Sema4D) capable of targeting receptors on platelets, B-cells, endothelial cells and tumor cells. However, its plasma levels and significance in cardiovascular disorders have not been reported.

Hypothesis

We hypothesized that plasma soluble Sema4D levels increases in patients with heart failure (HF) and that such an increase reflects T-cell and/or platelet activation.

Methods

We established an ELISA method and detected soluble Sema4D levels in human plasma, and evaluated the effect of estradiol on Sema4D shedding in platelets with collagen-induced aggregation. Percentage of Sema4Dhigh T cells, B cells and platelets in HF patients and healthy controls was detected with flow cytometry.

Results

In healthy controls, plasma Sema4D levels were higher in men than women (5.15 ± 3.30, n=63, vs. 4.19 ± 2.39 ng/mL, n=63, P<0.05), and cleavage of Sema4D in platelets activated by collagen was inhibited by estradiol. In HF patients, plasma Sema4D levels were significantly higher than those in healthy controls (8.94 ± 5.89 ng/mL, n=157 vs. 4.67 ± 2.99 ng/mL, n=126, P<0.0001) with the highest levels being in HF patients with diabetes mellitus (DM) (10.45 ± 5.76 ng/mL, n=40). We also found that there was a higher percentage of Sema4Dhigh CD3+ (P<0.01), CD4+ (P<0.001), and CD8+ (P<0.01) T-cells in samples from HF patients, but no change in B cells and platelet Sema4D expression levels.

Conclusions

Plasma Sema4D levels are increased in HF patients, especially in those who also have diabetes. There was an accompanying increase in the Sema4Dhigh population of T-cells, suggesting a potential role of these T-cells in heart failure.
Abstract 330: Ethnic Variability in the Association of Pulse Pressure with Adhesion Pathway Proteins: The Multi-Ethnic Study of Atherosclerosis (MESA) Study
Authors: Berardi Cecilia , Tsai Michael , Pankow James S , Sale Michele , de Andrade Mariza , Sicotte Hugues , Tang Weihong , Hanson Naomi , Decker Paul A , Kirsch Phillip S , Bielinski Suzette J
Published: July 2021

Pulse pressure (PP), defined as the difference between systolic and diastolic blood pressure, is considered a marker of large artery stiffness and has been shown to independently predict cardiovascular disease. Differences among ethnicities with regard to PP have been previously described, yet the underlying biological mechanisms are poorly understood. In addition, biomarkers of inflammation and matrix remodeling have been shown to be involved in the development of arterial stiffness. The aim of this study was to investigate the role of adhesion pathway components in the development of arterial stiffness measured by PP among four ethnicities. E-cadherin, HGF, L-selectin, P-selectin, 6-Ckine, MMP1, MMP2, RANTES, SDF1, SLPI, TGFβ1, TIMP2 and VCAM were measured in a random sample of 2402 participants (579 African Americans, 619 Caucasians, 600 Chinese and 604 Hispanics) in the Multi-Ethnic Study of Atherosclerosis (MESA) study at Exam 2. PP was measured concurrently and its association with each adhesion protein level was assessed via linear regression in each ethnicity. All analyses were adjusted for age, sex, and traditional cardiovascular risk factors. Mean PP was significantly different among ethnicities: 55.9±18.0 mmHg in African Americans, 52.1± 16.1 mmHg in Caucasians, 51.4±16.4 mmHg in Chinese, and 55.0±17.5 in Hispanics, p<0.001. MMP2 was positively associated with PP among African Americans (β=2.3 (0.6), p<0.001) and Caucasians (β=1.7 (0.5), p=0.001); TIMP2 among Caucasians (β=1.5 (0.5), p=0.002) and to a lesser extent among African Americans (β=1.2 (0.6) p=0.050) and Chinese (β=1.3 (0.6), p=0.034). SDF1 was associated to PP in African Americans (β=1.4 (0.6), p=0.017) and in Chinese (β=1.1 (0.5), p=0.042); finally an association existed between TGFβ1 and PP in C (β=1.1 (0.5), p=0.025). None of the studied proteins was associated with PP in Hispanics. In conclusion, components of the adhesion pathway were correlated to PP in this large multi-ethnic cohort, possibly giving new insight on the pathophysiology of arterial stiffness. In addition, heterogeneity by ethnicity suggests different mechanisms in the development of arterial stiffness.
Abstract 346: Platelet Reactivity to Aspirin and Clopidogrel in Patients with Acute ST Elevation Myocardial Infarction and its Correlation with High Sensitivity C- Reactive Protein
Authors: Rajdev Archana , Penciu Oana , Bradley Jacqueline , Mihu Cristina , Siqueros Alan , Petrini Joan , Wasserman Hal , Warshofsky Mark
Published: July 2021

INTRODUCTION

Implantation of bare metal or drug eluting stents supported by dual antiplatelet therapy (DAPT) is standard treatment for the management of patients with ST elevation myocardial infarction (STEMI). Individual response to aspirin and clopidogrel is heterogeneous, and decreased response is associated with thrombotic events following stenting. We postulated that systemic inflammation at the time of STEMI would diminish responsiveness to DAPT. The aim of this study is to evaluate the correlation between elevated high-sensitivity C-reactive protein (hs-CRP) as a marker of inflammation and decreased platelet sensitivity to DAPT in STEMI.

METHODS

We recruited patients with STEMI undergoing percutaneous coronary intervention (PCI) who received oral clopidogrel 600 mg loading dose followed by 75 mg daily maintenance dose and aspirin 325 mg daily. Platelet reactivity and hs-CRP were measured within 72 hours of PCI and at 6 weeks. For patients receiving eptifibatide, blood samples were taken 48 hours after discontinuation. Platelet reactivity was assessed using the VerifyNow platelet function analyzer. A cut-off value of 208 platelet reaction units (PRU) was used to define high on-clopidogrel platelet reactivity (HCPR) and a value of 454 aspirin reaction units (ARU) was used to define high on-aspirin platelet reactivity (HAPR).

RESULTS

In 20 patients aged 31 to 85, in hospital and 6 weeks after STEMI, hs-CRP was 6.7 (SD 4.0) and 2.6 (SD 3.2) respectively, p< 0.01. Changes in ARU from 408.3 (SD 54.3) to 425.2 (SD 68.2) and PRU from 157.8 (SD 74.7) to 164.2 (SD 75) were not statistically significant. 2 patients had HAPR in hospital; 1 became sensitive at follow up. 2 patients developed HAPR and HCPR. We saw a trend towards higher PRU in diabetic patients and those prescribed statins.

CONCLUSIONS

Although we found a significant difference in hs-CRP levels between the first and second time point, no significant difference was found in on-aspirin and on-clopidogrel platelet reactivity between the time points.Thus, in this small series, the acute inflammatory state associated with STEMI did not appear to influence the on-DAPT reactivity at the dosages used. Trends among those with diabetics and prescribed statins will be discussed
Abstract 393: Anti-inflammatory Properties of Aqueous Extracts of Sesame Oil
Authors: Selvarajan Krithika , Aluganti Narasimhulu Chandrakala , Bapputty Reena , Parthasarathy Sampath
Published: July 2021

Background

Dietary intervention to prevent atherosclerosis and inflammation has been a major focus in recent years. Sesame oil (SO), widely used in many Asian countries, has been reported to help reduce high blood pressure. It has also been shown to reduce plasma cholesterol, low density lipoprotein (LDL) cholesterol and triglyceride levels. We previously reported that SO was effective in inhibiting atherosclerosis in LDL-receptor negative mice. In this study we tested whether the aqueous, non-lipid components of SO might have anti-inflammatory effects.

Methods

Sesame oil was extracted using ethanol:water mixture, lyophilized and reconstituted in water. To study anti-inflammatory effect, RAW 264.7 cells (macrophage cell line) were treated with the aqueous extract in the presence or absence of lipopolysaccharide (LPS) for 24 hours. RNA was extracted using Trizol. mRNA expression of inflammatory cytokines such as IL-1α, IL-6 and TNF-α were analyzed by real time PCR. Protein expression was determined by western blot analysis. To identify the mechanism of action, we performed luciferase assay using HepG2-LXR reporter cell lines.

Results

LPS induced the expression of IL-1α, IL-6 and TNF-α mRNA levels in RAW cells. The extract alone did not significantly affect the expressions of inflammatory cytokine genes. However, when treated together with LPS, sesame oil aqueous extract inhibited the mRNA levels of these cytokines significantly. Treatment with LPS together with SO extract also decreased the protein expression of these cytokines. The SO extract induced LXR expression as identified by the luciferase assay system in HepG2-LXR reporter cells.

Conclusion

These findings suggest that the aqueous portion of SO might be effective in preventing inflammation. Furthermore, the activation of LXR might suggest additional effects on lipid metabolism. Identifying the specific components present in the aqueous extract will be instrumental in developing treatment modalities for atherosclerosis and other inflammatory conditions.
Abstract 413: Myeloperoxidase-mediated Methionine Oxidation Renders Apolipoprotein A-I Amyloidogenic
Authors: Jayaraman Shobini , Gantz Donald L , Chan Gary K , Witkowski Andrzej , Cavigiolio Giorgio
Published: July 2021

Background

Elevated plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, while dysfunctional apoA-I promotes atherosclerosis. The high concentration of myeloperoxidase (MPO) produced by activated macrophages in the atherosclerotic lesions generates an oxidative environment that impacts apoA-I and reduces its antiatherogenic functions.

Recent clinical studies revealed that amyloid deposition in the atherosclerotic plaques increases with aging. Interestingly, apoA-I was identified as a central component of these atherosclerosis-related amyloids.

Hypothesis

In this study, we tested the hypothesis that MPO-mediated oxidation of apoA-I can produce protein variants that are structurally destabilized and amyloidogenic.

Methods

Plasma apoA-I purified from normal individuals was exposed to chemical (hydrogen peroxide) and enzymatic (MPO-H
2
O
2
-chloride system) oxidation. Oxidized apoA-I was incubated at 37 °C and pH 6.0 to induce fibril formation.

Results

While no fibril formation was detected in intact apoA-I, both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after 12 h incubation at 37 °C. A large molar excess (~1000:1) of H
2
O
2
was needed to generate apoA-I fibrils, whereas exposure of apoA-I to a 1:1 oxidant:apoA-I molar ratio in the presence of MPO was sufficient to confer amyloidogenic properties to the protein. Analysis by gel electrophoresis, CD spectroscopy and mass spectrometry of soluble oxidized protein and of isolated fibrils indicate that no significant protein degradation occurred upon oxidation (both chemical and enzymatic) or fibril formation. The amyloid fibrils were composed of full-length apoA-I with all three native methionines converted to methionine sulfoxide.

Conclusions

In contrast to hereditary amyloidosis wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid fibril deposition in the atherosclerotic plaques
in vivo
.
Abstract 456: Comparative Effects of Angiotensinogen versus Renin Inhibition on Established Atherosclerosis and Obesity in Hypercholesterolemic Mice
Authors: Balakrishnan Anju , Howatt Deborah A , Lu Hong , Graham Mark J , Mullick Adam E , Crooke Rosanne M , Feldman David , Cassis Lisa A , Daugherty Alan
Published: July 2021

Background and Objective

The renin angiotensin system plays a critical role in atherosclerosis. Inhibition of angiotensinogen (AGT) or renin prevents atherosclerosis. AGT inhibition also ablates diet induced obesity. The purpose of this study was to compare the effects of AGT and renin inhibition on established atherosclerosis and obesity.

Methods and Results

To determine the role of AGT, male LDL receptor -/- mice were fed a saturated fat-enriched diet for 12 weeks to establish atherosclerosis (N=80). Body weight increased about 1 g/week in the 12-week duration. Twenty mice were then terminated to assess baseline atherosclerosis, and the remaining mice were randomly grouped to administer either control- or AGT-antisense oligonucleotides (ASO) (50 mg/kg/week ip; N=20/group) for 12 weeks. To determine the role of renin inhibition, male LDL receptor -/- mice were fed the same fat-enriched diet for 16 weeks (N=55). Mean body weight gain was about 1 g/week. Twenty-one mice were terminated to assess baseline atherosclerosis, and the remaining mice were randomly grouped to infuse either PBS (N=24) or aliskiren (12.5 mg/kg/d; N=10) for 12 weeks. Inhibition of AGT or renin significantly decreased systolic blood pressure. Atherosclerotic lesions were quantified in the aortic arch. Inhibition of either AGT or renin attenuated the rogression of atherosclerosis as compared to their relative controls (P=0.047 and P≤0.001, respectively), whereas neither mode of inhibition regressed atherosclerosis compared to their respective baseline. Renin inhibition did not influence the fat diet-induced body weight gain. Surprisingly, inhibition of AGT not only abolished body weight gain, but also led to pronounced loss of body weight (baseline versus 12-week injection of AGT-ASO: 36.0±0.8 versus 32.2±0.9 g; P<0.001). Echo MRI analyses demonstrated that the lower body weight in mice administered AGT-ASO was attributed to less fat mass, while lean mass was comparable between control-ASO and AGT-ASO groups.

Conclusions

Inhibition of either AGT or renin attenuated the continued development of atherosclerosis. Inhibition of AGT also profoundly reduced body weight. This highlights a disparity between the two modes of RAS inhibition.