bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 305: Cytokine Response to Concentrated Bone Marrow Aspirate Injections in Patients With Critical Limb Ischemia
Authors: Wang S. Keisin , Green Linden , Babbey Cliff , Motaganahalli Raghu , Kusumanchi Praveen , Tholpady Sunil , Fajardo Andres , Murphy Michael
Published: July 2021

Objective:
No medical intervention is approved for patients who suffer from critical limb ischemia (CLI) without a surgical revascularization option. Concentrated bone marrow aspirate (cBMA) injections have demonstrated safety and efficacy in increasing 1-year amputation-free survival (AFS) in the phase III multicenter, double blind, randomized controlled MOBILE trial. The response to cBMA injection is described herein.

Methods:
A murine (IL-2Rγ
-/-
) hind-limb ischemia model was employed to assay blood and tissue levels of angiogenic markers after MarrowStim
TM
derived cBMA injection. Responders to therapy were selected by cutaneous laser Doppler. Animals were sacrificed at various time points post-injection during which blood and distal limb tissue was harvested. 10 patients were enrolled into the MOBILE Continuing Access trial from May to December 2016 and received cBMA injections into the ischemic limb. Blood was collected at days 1, 3, 7, 14, 45, 60, and 90. Endothelial progenitor cells (EPCs) and protein markers of tissue ischemia were assayed by FACS and ELISA respectively.

Results:
In mice, cBMA produced a marked increase in gross tissue survival, capillary density, and perfusion compared to the control limb despite low engraftment of human cells. There was no inflammatory infiltration at any of the injection sites. Subanalysis of groups that had differing responses demonstrated crucial increases in FGF-2, VEGF, angiopoietin-2, IL-1β, and TNF-α. 7 subjects donated adequate of blood samples for FACS analysis; of this cohort, 5 patients had demonstrable increases in their EPC to non-EPC ratios immediately post-treatment. No statistically significant changes in FGF, VEGF, ANG1/2, PDGF, and GM-CSF was observed in the systemic circulation.

Conclusions:
cBMA has demonstrated efficacy in increasing 1-year AFS in CLI patients without surgical revascularization options. However, the response to treatment is variable and further studies are required to predict those who would benefit the most from cBMA.
Abstract 309: Blockade of Transforming Growth Factor Beta Activity in Elastase-Induced Aortic Injury in Mice Induces a Human-Like Abdominal Aneurysm
Authors: Clement Marc , Lareyre Fabien , Raffort Juliette , Pohlod Stefanie , Patel Meghana , Esposito Bruno , Master Leanne , Finigan Alison , Villabuena Pablo , Stamponi Marco , Taleb Soraya , Trashet Bram , Mallat Ziad
Published: July 2021

Background:
Abdominal aortic aneurysm (AAA) carries important morbidity and mortality and is resistant to medical therapy. Current experimental models do not accurately reproduce the major features of the human disease. There are 2 major categories of mouse models of AAA: those that induce medial dissection, which is not a major characteristic of human AAA, and those that induce aortic dilatation but are self-contained and do not progress to rupture.

Methods:
We hypothesized that blockade of TGFβ activity, a guardian of vascular integrity and immune homeostasis, and a major causal factor in genetically triggered thoracic aortic aneurysms in humans, would impair vascular healing in models of ‘non-dissecting’ abdominal aortic dilatation, and would lead to continuous aneurysmal growth until rupture. We tested this hypothesis in the elastase-induced abdominal aortic dilatation model in mice. We analyzed AAA development and progression using ultrasound in vivo, advanced synchrotron-based ultrahigh resolution imaging ex-vivo, and a combination of biological, histological and flow cytometry-based cellular and molecular approaches in vitro.

Results:
We show that systemic blockade of TGFβ activity using a neutralizing mouse monoclonal anti-TGFβ antibody induces a transition from a model of self-contained aortic dilatation to a model of sustained aneurysmal growth culminating in rupture. TGFβ blockade enhances leukocyte infiltration and extracellular matrix degradation, and leads to sustained aneurysmal aortic dilatation, associated with the formation of an intra-luminal thrombus infiltrated with neutrophils, as seen in the human disease. Persistent AAA growth throughout the duration of the experiment is associated with wall disruption without medial dissection, and culminates in fatal aortic wall rupture. Monocyte/macrophage depletion substantially limits AAA severity.

Conclusions:
Endogenous TGFβ activity is required for the resolution of elastase-induced aortic injury. We expect that this new model will improve our understanding of the pathophysiology of AAA, and will be useful to identify new therapeutic targets.
Abstract 311: The Long Non-Coding RNA H19 Regulates Experimental Abdominal Aortic Aneurysm Development and Progression
Authors: Li Yuhuang , Jin Hong , Busch Albert , Chernogubova Ekaterina , Bäcklund Alexandra , Olofsson Anna , Eken Suzanne , Sun Changyan , Simon Nancy , Korzunowicz Greg , Eriksson Per , Hultgren Rebecka , Roy Joy , Spin Joshua , Tsao Philip , Maegdefessel Lars
Published: July 2021

Background:
Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA H19 as a novel regulator in abdominal aortic aneurysm development.

Methods and Results:
We profiled RNA transcript expression in two murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in ApoE-/- mice and porcine pancreatic elastase (PPE) instillation in C57BL/6 wildtype mice. The lncRNA H19 was identified as one of the most highly up-regulated transcripts in both mouse aneurysm models compared to sham-operated controls. This was confirmed by qRT-PCR and
in situ
hybridization. Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs)
in vivo
, significantly limited aneurysm growth in both models. Upregulated H19 correlated well with smooth muscle cell (SMC) content and its apoptosis in progressing aneurysms. Of importance, a similar pattern could be observed in human AAA tissue samples, and in a novel preclinical LDLR-/- Yucatan mini-pig aneurysm model.
In vitro
knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, while overexpression of H19 had the opposite effect. Interestingly, H19-dependent cell fate decisions in SMCs appeared independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array and proteomic analysis revealed the hypoxia-inducible factor 1-alpha (HIF1A) and interleukin 1 receptor-like 2 (IL1RL2) as the main downstream effectors being regulated via H19.

Conclusion:
The lncRNA H19 is a novel regulator of SMC survival and aortic inflammation in AAA development and progression. Ultimately inhibition of H19 expression might serve as a novel molecular therapeutic target for aneurysm disease.

Key words:
Long noncoding RNAs (lncRNAs); abdominal aortic aneurysm; smooth muscle cells (SMCs); Inflammation
Abstract 312: Finite Element Analysis-Derived Biomechanical Rupture Risk Index is Associated With Extracellular Matrix and Structure Organization in the Tunica Media of Abdominal Aortic Aneurysms
Authors: Lindquist Liljeqvist Moritz , Siika Antti , Kronqvist Malin , Gasser T. Christian , Eriksson Per , Hultgren Rebecka , Roy Joy
Published: July 2021

Objective:
An abdominal aortic aneurysm (AAA) ruptures if the vessel wall stress exceeds its strength, with high mortality as a result. The effect of biomechanical load on AAA gene expression is currently not well understood.

Methods:
Aortic tissue samples from 64 patients with an AAA that had been imaged with computed tomography angiography (CTA) before surgery were collected from the Stockholm AAA Biobank (STAAAB), separated into intima/media and adventitia wall layers and analyzed with Affymetrix HTA 2.0 microarrays. The aortas were segmented from the CTAs into three-dimensional digital models, which were then subjected to finite element analysis (FEA) without informing the software (VASCOPS A4clinics Research Edition) about age, gender, family history or blood pressure. Peak wall rupture index (PWRI), derived from the FEA, was used as a marker of biomechanical rupture risk. Differential gene expression, adjusted for age and gender, was studied with R. Enriched gene ontology processes were examined for annotated transcripts with GOrilla and REVIGO online tools.

Results:
No transcript was significantly associated with PWRI after 5% false discovery rate correction was performed, in either media nor adventitia. However, there were 1203 and 872 transcripts associated with PWRI at a nominal p-value of < 0.05 in media and adventitia, respectively. Analysis of enriched biological processes using the entire annotated transcript list ranked by fold-change revealed that extracellular matrix and structure organization in the media and nucleic acid metabolism and RNA metabolism in the adventitia, were the most enriched biological processes in aneurysms with high PWRI.

Conclusion:
These results suggest that biomechanical rupture risk associates with expression of extracellular matrix genes as well as the level of bulk gene activity.
Abstract 313: Protective Roles of Small GTP-Binding Protein GDP Dissociation Stimulator Against Angiotensin II-Induced Thoracic Aortic Aneurysm Formation and Rupture in Mice -A Possible Novel Therapeutic Target
Authors: Nogi Masamichi , Satoh Kimio , Kudo Shun , Omura Junichi , Kikuchi Nobuhiro , Satoh Taijyu , Sunamura Shinichiro , Kurosawa Ryo , Ohtsuki Tohmohiro , Numano Kazuhiko , Suzuki Kota , Shimokawa Hiroaki
Published: July 2021

Background:
Statins reduce the incidence and development of thoracic aortic aneurysm (TAA) and rupture. We have previously identified that small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins.

Methods and Results:
To examine the role of SmgGDS in TAA formation, Apoe
-/-
and Apoe
-/-
SmgGDS
+/-
(DKO) mice were infused with angiotensin II (AngII, 1,000 ng/min/kg) for 4 weeks. There was no significant difference in blood pressure between the 2 genotypes in response to the AngII treatment. However, during the follow-up, 36% of DKO mice died suddenly due to TAA rupture, whereas there was no TAA rupture in Apoe
-/-
mice (
P
<0.05,
n
=14 each). Histological analysis of DKO mice showed dissections of major thoracic aorta in the early phase of AngII infusion (day 3~5). We performed ultrasound imaging every week to follow the serial changes in aortic diameters. Diameter of the ascending aorta was progressively and significantly increased in DKO mice compared with Apoe
-/-
mice (1.64±0.06 vs. 1.43±0.05 mm at 4 weeks,
P
<0.05,
n
=14 each), whereas that of the abdominal aorta was comparable between the 2 genotypes. Indeed, there was no significant difference in the incidence of AngII-induced abdominal aortic aneurysm (AAA) formation between the 2 genotypes (both 75%). Western blotting demonstrated that AngII-induced activations of JNK and ERK were significantly higher in the thoracic aorta of DKO mice compared with Apoe
-/-
mice (
P
<0.01,
n
=6 each). For mechanistic analyses, we primary cultured aortic smooth muscle cells (AoSMCs) from the 2 genotypes. After AngII (100 nM) treatment for 24 hours, DKO AoSMCs showed significantly increased JNK activity, cyclophilin A secretion, and oxidative stress levels compared with Apoe
-/-
AoSMCs (
P
<0.01,
n
=6 each). Interestingly, AngII-induced upregulation of Nrf2, a master regulator of cellular responses against environmental stresses, was significantly less in DKO AoSMCs compared with Apoe
-/-
AoSMCs (
P
<0.01,
n
=6). Finally, expressions of matrix metalloproteinase-2 and -9 were significantly increased in DKO AoSMCs compared with Apoe
-/-
AoSMCs.

Conclusions:
These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.
Abstract 315: Calcium Dose Dependently Influences Endothelial Cell Angiogenesis
Authors: Mulangala Jocelyne , Akers Emma J , Psaltis Peter J , Nicholls Stephen J , Di Bartolo Belinda A
Published: July 2021

Background:
Peripheral artery disease (PAD) is a progressive occlusive disease of the arteries and a vascular complication in diabetes. Vascular calcification (VC) is implicated as a potential driver of PAD, and although the exact mechanisms are unclear, the site and location of calcification within the arterial wall contributes greatly. Long considered a passive process, VC is now recognised as a tightly regulated active process balancing the promotion and inhibition of calcification in the arterial wall. There is little evidence however, to demonstrate the effect of calcification on endothelial cell angiogenesis. This study sought to investigate the effects of calcium as a known inducer of calcification on
in vitro
angiogenesis.

Methods:
Human Coronary Artery Endothelial Cells were cultured and treated with increasing calcium concentrations (CaCl
2
2.45-3.3 mM) for 24h. Proliferation, migration and tubule formation assays were conducted and real-time PCR assessed angiogenic and osteogenic genes. Alkaline phosphotase (ALP) activity was measured in supernatants following treatment.

Results:
High concentrations of calcium reduced cell proliferation with a corresponding increase in ALP production suggesting release of osteogenic stimuli adversely affects cell viability. Mid-range concentrations of calcium induced a significant increase in cell migration (1.0 vs 2.4±0.3, p<0.05) while higher concentrations elicited no effect. Calcium treatment demonstrated a dose response where mid-range concentrations increased gene expression of hypoxia-inducible factor-1α (>500 fold), and fibroblast growth factor-2 (>150 fold). This increase corresponded with a decrease (1.0 vs 15.02±4.24; p<0.0001) in osteoprotegerin (OPG) at mid-range calcium with a significant increase at the highest concentration (1.0 vs 342±13.27; p<0.01) illustrating calcium-induced expression of OPG, a known protective gene in VC, may also regulate angiogenesis.

Conclusion:
This is the first demonstration investigating the effects of calcium on endothelial cell angiogenesis. These findings suggest that calcium can directly affect genes involved in regulating angiogenesis, and could therefore provide an opportunity to develop potential treatments.
Abstract 318: Modulation of Neointimal Hyperplasia Severity in Rats by Commensal Microbial Transfer
Authors: Cason Cori A , Kuntz Thomas M , Gottel Neil , Xiong Liqun , Jiang Qun , Chang Eugene B , Gilbert Jack A , Ho Karen J
Published: July 2021

Neointimal hyperplasia is a major contributor to restenosis after arterial interventions. The genetic and environmental mechanisms underlying the variable propensity for neointimal hyperplasia between individuals are not well understood. One possible modulator could be commensal gut microbes. To address whether microbes mediate neointimal hyperplasia, we cohoused genetically different rats (Lewis [LE] and Sprague-Dawley [SD]) which harbor different commensal microbes and compared neointimal hyperplasia 2 weeks after carotid angioplasty in the cohoused and non-cohoused cohorts. Cohousing is a means of microbial transfer between cage inhabitants. We observed that differences in neointimal hyperplasia between non-cohoused LE and SD rats (median intima+media [I+M] area 0.12 mm
2
LE vs. 0.26 mm
2
SD, P<.0001;Mann-Whitney) were mitigated when rats are cohoused for 1 month (Figure 1A), suggesting an environmental effect that outweighs the genetic influence. Specifically, I+M area decreased by 23% in SD rats that were cohoused with LE rats (P<.0001;Mann-Whitney), and there was a trend towards a 10% increase in I+M area in cohoused LE rats. To identify specific bacteria associated with the change in neointimal hyperplasia, we monitored fecal bacteria over time using 16S rRNA sequencing. Principal component analysis revealed that fecal samples from cohoused rats diverged from non-cohoused rats in both strains (P<.001 SD, P=.008 LE;PERMANOVA) (Figure 1B). The greatest change was cohoused SD samples becoming similar to non-cohoused LE samples over time, which correlates with the carotid morphometric data. Comparative analysis showed that abundance of the bacterial genera Peptococcus and Blautia negatively correlated with I+M area in both strains (P<.001;Fisher z transform, Bonferroni corrected, Spearman’s ρ -0.8 for both). Ongoing studies will further delineate the potential causative relationship between these microbes and neointimal hyperplasia.
Abstract 319: Effects of Muscle Stretching on Flow-Mediated Dilation of Popliteal Artery in Patients With Peripheral Artery Disease
Authors: Hotta Kazuki , Batchelor Wayne B , Graven James , Dahya Vishal , Noel Thomas E , Ghai Akash , Katopodis John N , Dixon William C , Chheda Jegghna , Liberatore Lia , Behnke Bradley J , Muller-Delp Judy M
Published: July 2021

Patients with peripheral artery disease (PAD) frequently have walking impairment due to lower extremity claudication. Our preliminary results in a rat model of aging indicate that a program of daily calf muscle stretching improves endothelium-dependent dilation of soleus muscle arterioles and increases soleus muscle blood flow during exercise. However, the effects of muscle stretching on the function of arteries supplying the legs of PAD patients is unknown. We hypothesized that daily calf muscle stretching improves vascular endothelial function and walking distance in PAD patients. To test our hypothesis, a randomized, non-blinded, crossover study was performed. Four weeks of muscle stretching (30 min/d, 5 days/wk) and 4 weeks of sedentary lifestyle (no stretching) were performed in random order. Thirteen patients with PAD participated in this study (71 ± 2 years old; 7 males and 6 females). During the stretching intervention both ankle joints were maintained at 15o of dorsiflexion using ankle dorsiflexion splints to stretch their calf muscles at home. Flow-mediated dilation (FMD; dilation to post-occlusion reactive hyperemia) and nitroglycerin-induced dilation (dilation to sublingual 0.4 mg nitroglycerin) of the popliteal artery were measured after 4 weeks of muscle stretching and after the no stretching period using ultrasound. A six-minute walk test was also performed to obtain walking distance. After 4 weeks of muscle stretching, FMD and 6-minute walking distance significantly improved as compared to the values measured after 4 weeks of no stretching (FMD: 5.2 ± 0.6 % vs. 3.7 ± 0.4 %, P=0.003 stretching vs. no stretching, 6-minute walking distance: 355 ± 32 m vs. 311 ± 31 m, P=0.007, stretching vs. no stretching; mean ± SE). No difference in nitroglycerin-induced dilation was found between groups (10.9 ± 1.4 vs. 9.9 ± 1.1 %, P=0.54, stretching vs. no stretching). Percentage change of walking distance (%change = [(stretching - no stretching) / no stretching] x 100) significantly correlated with the %change of FMD (R
2
=0.65, P=0.03). These results indicate that static calf muscle stretching enhances vascular endothelial function of the popliteal artery, contributing to improvement of walking tolerance in PAD patients.
Abstract 320: Delivery of Hepatocyte Growth Factor mRNA From Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease
Authors: Huang Ngan F , Hou Luqia , Alcazar Cynthia , Strassberg Zachary , Hopkins Michael , Zaitseva Tatiana , Yakubov Eduard , Paukshto Michael V
Published: July 2021

Biological approaches to augment angiogenesis are promising for treatment of peripheral arterial disease (PAD). We propose the use of scaffold-based modified mRNA (mmRNA) delivery as a favorable approach for transient, localized gene delivery. We hypothesized that hepatocyte growth factor (HGF) mmRNA-seeded nanofibrillar scaffolds will enable localized and temporally controlled delivery of mmRNA, leading to augmentation of angiogenesis in a murine model of PAD. To establish the efficacy of mmRNA therapy, mmRNA encoding green fluorescence protein (GFP) was used as a fluorescent reporter for quantification of transfection efficiency. Aligned nanofibrillar collagen scaffolds were loaded with mmRNA and lipofectamine transfection agent. The temporal kinetics of mmRNA release into media was measured by ribogreen assay. To determine the transfection efficiency, human fibroblasts were cultured on the aligned nanofibrillar scaffolds, or on tissue culture plastic, and the efficiency of transfection was measured for up to 7 days and assayed for GFP expression. Based on ribogreen assay, the cumulative release of GFP mmRNA over the course of 14 days was 235 ng/cm scaffold.
In vitro
transfection efficiency on aligned scaffolds (75%) was markedly higher than on tissue culture plastic (45%) after 24h. The persistence of cellular transfection as quantified by western blotting showed GFP expression >5 days post-transfection. Next, to demonstrate therapeutic efficacy for treatment of PAD, scaffolds releasing HGF or GFP mmRNA were transplanted to the site of the murine ischemic hindlimb. At the end of the 14 day experiment, laser Doppler spectroscopy showed that HGF mmRNA scaffold group had a higher mean perfusion ratio (0.32 ±0.10) than the GFP mmRNA scaffold group (0.23±0.14), suggesting that HGF-scaffolds improved blood perfusion. In summary, these data suggest that HGF mmRNA-releasing scaffolds marked improved blood perfusion in a murine model of PAD.
Abstract 321: Central Blood Pressure Regulation in Relation to Hypertension and Adiposity in Youth
Authors: Hultgren Neil E , Evanoff Nicholas G , Kaizer Alexander M , Rudser Kyle D , Dengel Donald R , Kelly Aaron S , Ryder Justin R
Published: July 2021

Introduction:
The relationship between different central BP measurements with hypertension status in youth is not well documented. We hypothesized that measures of central BP would be positively associated with hypertension status in youth independent of adiposity.

Methods:
We recruited 149 males and 160 females for this cross-sectional analysis (mean±SD: age = 12.8±2.7; BMI percentile (%) = 78.5±27.8). Body fat % was measured by dual energy X-ray absorptiometry (DXA) and brachial BP was measured using an automated cuff to calculate systolic BP (SBP) and diastolic BP (DBP). Determined by systolic percentile, there were 238 normotensive (<90
th
), 29 pre-hypertensive (<90
th
- <95
th
), and 42 hypertensive (<95
th
) individuals enrolled. Central BP was determined using the SphygmoCor MM3 system to calculate carotid-aorta SBP (caSBP) carotid-aorta DBP (caDBP) radial-aorta SBP (raSBP) and radial-aorta DBP (raDBP).

Central BP measures were compared across hypertension status groups using ANCOVA, with post-hoc Tukey HSD, adjusted for age, sex, and race. Pearson correlations (unadjusted) and multiple linear regression models, examining the relationship between central BP measures with brachial BP adjusted for age, sex, race, and height, were conducted with further adjustment for body fat % (shown as β±SE).

Results:
raSBP, caSBP, raDBP, and caDBP were significantly different between the normotensive and hypertensive groups (all p<0.001). No statistically significant differences were found between normotensive and pre-hypertensive or between pre-hypertensive and hypertensive groups. raSBP and caSBP were correlated to SBP (r=0.59, r=0.62, respectively, p<0.001). raDBP and caDBP were correlated to DBP (r=0.58, r=0.6, respectively, p<0.001). In regression analysis, SBP was positively associated with both raSBP and caSBP (β=0.3±0.06, p<0.001) and (β=0.28±0.08, p<0.001), respectively. DBP was positively associated with raDBP and caDBP (β=0.31±0.06, p<0.001) and (β=0.31 ±0.06, p<0.001), respectively. All associations remained significant after adjustment for body fat %.

Conclusion:
These data suggest that central BP, regardless of measurement site, is highly associated with brachial BP and hypertension status in youth independent of adiposity.
Abstract 325: Usefulness of Peripheral Vascular Disease Assessment on Readmission in a Heart Failure Program Cohort
Authors: Lee Hong Seok , Pekler Gerald , Visco Fernand , Savadkar Amrut
Published: July 2021

Background:
Peripheral arterial disease (PAD) was reported to have a relationship with functional capacity in heart failure patients. Heart failure patients presenting with a good functional capacity have been found to have better metabolic equivalents (METs). Recognizing and managing patients’ functional capacity surrogates like ankle-brachial index (ABI) and METs will be beneficial to improve the rate of readmission, however, little is known about the relationship with readmission rate in heart failure patients. We assessed readmission rate within 30 days after discharge using functional capacity assessment.

Methods:
860 patients who were followed in the cardiology clinic from 2005 to 2015 were included. We analyzed the 240 patients who were admitted with a diagnosis of acute heart failure. Patients who were unable to cardiac rehabilitation or who had severe lung disease were excluded. Heart failure is classified as a reduced ejection fraction (HFrEF, EF <40) and preserved ejection fraction (HFpEF, EF>=50). MET (Metabolic equivalents) level was used for functional capacity. If ABI was less than 0.9 or over 1.4, patients were regarded to have peripheral arterial disease.We found no significant difference between our HFpEF and HFrEF patients (Mean METS 7.5±0.6 vs 7.4±0.7). ABI did not show any significant difference (1.1±0.2 vs 1.0±0.3). In multiple logistic regression analyses, HFpEF patients with more than 4 MET level were likely to have fewer readmissions rate compared to HFpEF patients with less than 4 METS level [odds ratio (OR): 0.54, confidence interval (CI): 0.35-0.81]. HFpEF patients with ABI between 0.9 and 1.4 had less readmission rate compared to HFpEF with less than 0.9 or more than 1.4 ABI [OR: 0.62, CI: 0.41-0.77] after related risk factors adjustment.

Conclusion:
In conclusion, good functional status with better METs and ABI in HFpEF patients was significantly associated with less readmission rate. ABI might be a surrogate factor for assessing functional capacity in HFpEF patients. This result implies that heart failure patients’ functional capacity might need to be assessed to decrease readmission rate.
Abstract 327: Blood Vessels Need Ras Signaling to Maintain the Structure Integrity
Authors: Hu Wenquan , Rana Ujala , Liu Zhong , Zhao Baofeng , Kumar Suresh , North Paula , Miao Qing (Robert)
Published: July 2021

The localization of prenylated Ras at the plasma membrane promotes activation of Ras by receptor tyrosine kinases, such as VEGF and FGF receptors. Although Ras has been implicated in angiogenesis, the exact regulatory mechanisms controlling Ras translocation and activation are currently unclear because little is known regarding molecules that control Ras translocation. Nogo-B receptor (NgBR) was identified as a receptor specific for Nogo-B, a cell surface ligand involved in blood vessel remodeling. Our recent study demonstrated that NgBR has a conserved hydrophobic pocket that promotes the membrane accumulation of Ras by directly binding prenylated Ras at the plasma membrane. As we expected, NgBR knockdown in endothelial cells diminishes the membrane localization of Ras and consequently abolishes VEGF/FGF-stimulated activation of Ras and Ras-mediated signalings such as phosphorylation of Akt and ERK. Therefore, NgBR knockout mouse is a unique animal model for examining the effects of Ras plasma membrane localization and Ras signaling on the morphogenesis of endothelial cells. Genetic deletion of NgBR in endothelial cells resulted in embryonic lethality and dilated cerebral blood vessels with fewer pericytes, which resembles the vascular lesion happened in cerebral cavernous malformation (CCM). CCM is characterized by an abnormal cluster of enlarged blood vessels in the brain and spinal cord and caused by dysfunction of three CCM genes (CCM1/2/3), which are required for maintaining endothelial cell (EC) junctions and pericyte recruitment. Our studies showed that NgBR transcript levels decrease in human CCM lesion, and NgBR endothelial specific knockout in mice results in decreased transcription of CCM1/2 in the yolk sac. Additional support for NgBR-CCM1/2 connections comes from studies using cultured human brain microvascular ECs, where loss of NgBR expression also decreases CCM1/2 transcription via NgBR-mediated Ras pathway, which is required for the expression of key transcription factors that are involved in regulating transcription of CCM1/2 genes. Our findings suggest that NgBR-Ras signaling pathway regulates CCM1/2 expression, and that disrupting this signaling pathway results in cerebrovascular malformation.
Abstract 328: Coagulation Proteases are Potential Regulators in the Development of Exercise-Related Endofibrosis
Authors: Posthuma Jelle J , Posma Jens J , Schep Goof , Bender Mart , van Oerle Rene , ten Cate Hugo , Spronk Henri M
Published: July 2021

Background:
High performance athletes can develop symptomatic arterial flow restriction during exercise, caused by endofibrosis. The disease is an poorly understood condition, characterized by fibrosis of the external iliac artery. Pathophysiological processes involved in the development of endofibrosis are unknown. However, evidence shows that coagulation enzymes such as thrombin and factor Xa (FXa) can influence pro-fibrotic processes, mainly mediated through activation of protease activated receptors (PAR).

Aim:
We investigated the immunohistochemical characteristics of endofibrotic lesions to determine the potential role of coagulation proteases and their receptors in development of endofibrosis.

Methods:
19 arterial endofibrotic specimens were collected during endarterectomy. As controls 20 arterial segments of the external iliac artery were collected post mortem from individuals who donated their body to medical science, with no medical history of cardiovascular disease. Acquired arteries were paraffinized and cut in tissue sections for immunohistochemical staining. Data were analyzed using a Mann-Whitney U test. A 2-tailed p<0.05 was considered as statistically significant.

Results:
Endofibrotic segments contained a neo-intima, resulting in an intraluminal stenosis (42±14%). Compared to the intima of controls, endofibrotic lesions were highly positive for collagen (47±16% vs 15±5%, p=0.0001) and elastin (38±9% vs 16±4%, p=0.0003). These findings were accompanied by significantly increased alpha smooth muscle actin expression (51±10% vs 36±11%, p=0.01), which morphologically appeared to be myofibroblasts in endofibrotic lesions; which were hardly present in controls. In addition, PAR1 (49±17% vs 22±10%, p=0.0003) and PAR4 (39±8% vs 10±4%, p<0.0001) were upregulated and proenzymes of their activators, prothrombin and factor X were abundantly present in endofibrosis.

Conclusion:
This is the first study to show that myofibroblasts, and the subsequent collagen and elastin production, might be key factors in the development of endofibrosis. The special association suggests that these processes may be regulated through activation of PARs by coagulation proteases, which needs to be established in further studies.
Abstract 329: ST266 Attenuates Neointima Formation After Arterial Balloon Angioplasty in Rats
Authors: Rahimpour Sina , Wang Xuening , Liu Xiaoyan , Muratoglu Selen C , Banas Richard , Pham Si M
Published: July 2021

Objective:
Post-angioplasty restenosis due to neointima formation has been attributed to the inflammatory response after acute endoluminal injury. ST266 (Noveome Biotherapeutics, Inc.) a novel secretome derived from proprietary GMP-cultured human Amnion-Derived Multipotent Progenitor (AMP) cells, has been shown to be anti-inflammatory and to promote wound healing. This study examined the therapeutic potential of ST266 in a rat arterial balloon angioplasty model.

Methods:
Animals were randomly divided in the following groups (N=7): no-treatment (noTx), systemic ST266, systemic AMPs and local AMP implants. Neointima hyperplasia was induced in the iliac artery of Sprague-Dawley male rats using a 2F Fogarty embolectomy catheter. After surgery, animals in ST266 groups received 0.1, 0.5 or 1ml IV ST266 q.d. In the systemic AMP groups, single-dose (SD) of 0.5 million (M) or 1 M AMPs was injected via Inferior Vena Cava after the angioplasty. In AMP implant experiment 1 M, 5 M or 20 M AMPs were implanted in 300 μL Matrigel (MTG) around the iliac artery after balloon angioplasty. 28 days after the surgery, the iliac arteries were removed for histologic analysis. Re-endothelialization index was measured 10 days after balloon angioplasty.

Results:
1ml ST266 decreased Neointima/Neointima+Media ratio (N/NM) compared to noTx group (0.34±0.01 vs 0.54±0.04 resp.;
p
=0.004). ST266 also decreased Luminal Stenosis (LS) compared to noTx group (18.18±1.86 vs 39.23±5.75%;
p
=0.008). SD 1 M AMPs decreased LS compared to noTx group (39.23±5.75 vs 19.50±5.35%;
p
=0.033). Significant differences in N/NM were found between 20 M implanted AMPs and noTx group (0.35±0.02 vs 0.54±0.04 resp.;
p
=0.003) and the MTG-only group (0.53±0.05,
p
=0.007). 20 M implanted AMPs decreased the LS (16.78±2.47%) compared to both noTx (39.23±5.75%,
p
=0.001) and MTG-only groups (37.51±8.55%,
p
=0.016). 1ml ST266 significantly increased the re-endothelialization index 10 days after balloon angioplasty compared to noTx group (0.40±0.04 vs 0.14±0.037% resp.,
p
=0.002).

Conclusion:
ST266 reduces neointima formation and luminal stenosis and increases the re-endothelialization index after balloon angioplasty. Further research to elucidate the underlying mechanisms is ongoing.
Abstract 33: Activated Protein C Light Chain Provides an Extended Binding Surface for Anticoagulant Cofactor Protein S
Authors: Fernandez Jose A , Xu Xiao , Sinha Ranjeet K , Mosnier Laurent O , Griffin John H
Published: July 2021

Plasma protein S whose deficiency is linked to increased risk for thrombosis provides anticoagulant cofactor activity for activated protein C (APC) by enhancing rates of inactivation of factors Va and VIIIa. Previous APC mutagenesis studies showed that residues 35-39 in the gamma-carboxyglutamic acid domain are required for normal interactions with protein S, indicating the APC Gla domain binds protein S. Here we used mutagenesis of APC to interrogate the surface of APC’s light chain to identify the extended binding surface for protein S. We characterized the ability of protein S to enhance the anticoagulant activity of multiple recombinant APC variants using factor Xa-1-stage clotting assays using normal pooled plasma and protein S-depleted plasma. Mutations of residues L38, K43, I73, F95, and W115 in APC significantly reduced protein S’s cofactor activity. An APC variant carrying all of these five mutations lost all of protein S cofactor activity. On the crystallographic structure of APC, these five residues delineate an extended surface on only one side of the APC light chain that identifies the putative protein S binding site which is found on a face that is opposite APC’s catalytic triad site. Each of the APC variants with single or multiple L38, K43, I73, F95, and W115 mutations showed a normal ability to cleave SEAP-labeled PAR1 at Arg 41 and Arg 46, implying that the protein S-binding surface does not bind EPCR or PAR1. In summary, mutagenesis studies identify an extended surface on a single face of APC’s light chain for binding protein S. This knowledge will enable design and interpretation of new APC biologics with enhanced translational value.
Abstract 334: Pulmonary Embolism Response Team: Implementation and Outcomes at Northshore University Healthsystems
Authors: Iftikhar Omer , Smart Keith , Ahsan Alya , Tafur Alfonso
Published: July 2021

Introduction:
Pulmonary Embolism Response Team (PERT) protocols are used to expedite risk stratification and management of complex acute pulmonary embolisms (PE). The process varies by institution.

Objective:
To quantify mortality outcomes after implementing PERT at our practice (Figure 1).

Methods:
58 patients in our PERT registry were analyzed for parameters seen on table 1. Quantitative data is presented as median and qualitative data as percentages.

Results:
Median age was 70.5, with female predominance 65.5% (38 out of 58), PE severity index (PESI) of 101.6, and cancer in 33% (19 out of 58) [Table 1]. 10%(6 out of 58) received thrombolysis and 14%(8 out of 58) received IVC filter. 5 deaths occurred of which 60% (3 patients) were due to cancer progression.

Conclusion:
Our PERT algorithm is anchored in right ventricle to left ventricle (RV/LV) ratio on CT reading and does successfully select high risk PE patients. Mortality is similar to that expected by PESI stratification. Prospective comparative evaluation of PERT is needed to see if it affects early mortality.
Abstract 340: Drug-Eluting Balloon Versus Everolimus-Eluting Stent for Restenosis in a Bare-Metal Stent: A Meta-Analysis of Randomized Trials
Authors: Akinjero Akintunde M , Adegbala Oluwole , Akinyemiju Tomi
Published: July 2021

Background:
In-stent restenosis accounts for major morbidity and mortality among patients treated with Bare-Metal Stents (BMS). Early efforts to treat BMS in-stent restenosis with plain balloon angioplasty and first generation drug eluting stents (DES) have been ineffective, leaving drug-eluting balloon (DEB) and second generation DES, such as everolimus eluting stents (EES), as the only remaining options. For BMS in-stent restenosis, studies performed so far have yielded conflicting results, while prior meta-analyses have been influenced by inclusion of observational studies. This is the first meta-analysis to compare EES versus DEB using results from only randomized controlled trials (RCTs).

Methods:
A systematic search of PUBMED and EMBASE databases was conducted from first available date to August, 2016 for RCTs comparing DEB with EES. Two reviewers evaluated studies for eligibility and extracted data with binary restenosis rate as the main endpoint. We identified 901 unique citations. Odds ratios were pooled using random-effects modeling. Funnel plots were used to assess publication bias. Heterogeneity was assessed using I
2
statistic. All analysis were performed using Review Manager (RevMan) version 5.3 (Cochrane Collaboration, 2014).

Results:
Three RCTs met study eligibility criteria, with 684 patients and a mean follow-up of 9.5 months. There were 184 and 185 patients in the EES and DEB arms respectively. In pooled analyses, EES was not superior to DEB in binary restenosis rates (pooled odds ratio: 0.76; 95% confidence interval: 0.25-2.32; P=0.14). Heterogeneity was minimal (I
2
= 49%), and the funnel plot did not suggest publication bias.

Conclusion:
In patients with BMS in-stent restenosis, there were no significant differences in binary restenosis rates between EES and DEB. Our results can enhance physician decision-making regarding choice of revascularization tool in this patient population.
Abstract 342: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow
Authors: Zilberman-Rudenko Jevgenia , Reitsma Stephanie E , Wiesenekker Chantal P , Puy Cristina , Smith Stephanie A , Tucker Erik I , Travers Richard J , Maas Coen , Urbanus Rolf T , Gailani David , Morrissey James H , Gruber Andras , McCarty Owen J
Published: July 2021

Background:
Terminal complications of bacterial sepsis include development of consumptive coagulopathy referred to as disseminated intravascular coagulation. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway is capable of promoting thrombin generation and platelet activation and consumption in whole blood distal to thrombus formation under shear
ex vivo
and
in vivo
.

Aim:
Test the hypothesis that the presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner.

Methods and Results:
Presence of long-chain polyP in whole blood promoted platelet aggregation on immobilized collagen surfaces under shear flow. Long-chain polyP enhanced fibrin formation and shortened clotting times of plasma and whole blood. The addition of long-chain polyP promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow. Distal to the sites of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of the coagulation pathway reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation.

Conclusions:
This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could exaggerate sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.
Abstract 346: CD39/CD73 Expressing Exosomes Protect Against Arterial Thrombosis
Authors: Covarrubias Roman , Snoek Susanne A , Zhong Lin , Broekstra Niels , Finn Jonathan D , Gumina Richard J
Published: July 2021

Objective:
To determine whether expression of CD39/CD73 in exosomes protects against ferric chloride-(FeCl
3
) induced arterial thrombosis.

Approach and Results:
Ectonucleotidase triphosphate diphosphohydrolase-1 (CD39) and ecto-5’-nucleotidase (CD73) sequentially hydrolyze extracellular ATP or ADP to AMP and AMP to adenosine. We have previously shown that mice with increased CD39 expression are protected against arterial thrombosis. Specifically, increased CD39 expression on circulating monocytes protects against arterial thrombosis. Based on this previous work, we hypothesized that exosomes expressing CD39/CD73 could confer protection against arterial thrombosis and extend the time to arterial occlusion following FeCl
3
injury. To test this hypothesis, we stably transfected 293 T cells with mouse CD39 and CD73 or control plasmids then isolated exosomes from the supernatant. Exosomes isolated from mCD39/mCD73 transfected cells showed expression of mCD39 (6.60 μg/ml) and mCD73 (10.8 μg/ml) with a specific activity of 512,936 (U/μg) and 67,406 (U/μg) respectively as shown by phosphate hydrolysis assay (malachite green). To test whether mCD39/mCD73 exosomes protect against arterial thrombosis we infused 9.8 x 10
11
CD39/CD73 exosome particles, or control particles, into the jugular vein of wild-type mice 15 minutes prior to FeCl
3
injury of the carotid artery. Infusion of mCD39/mCD73 exosomes significantly increased the time to thrombosis when compared to control exosomes in wild-type mice (Figure; control exosomes: 545±15.67 seconds, n=4. mCD39/mCD73 exosomes: 1644±398.3 seconds, n=4.
P
=0.029).

Conclusion:
CD39/CD73 expressing exosomes protects mice against arterial thrombosis.
Abstract 354: Clots Are Potent Triggers of Inflammatory Cell Gene Expression: Indications for Timely Fibrinolysis
Authors: Campbell Robert A , Vieira-de-Abreu Adriana , Rowley Jesse W , Franks Zechariah G , Rondina Matthew T , Kraiss Larry W , Majersik Jennifer J , Zimmerman Guy A , Weyrich Andrew
Published: July 2021

Objective:
Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine if clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases.

Approach and Results:
We performed Next Generation RNA sequencing on monocytes extracted from whole blood clots. Thousands of mRNAs were differentially expressed by monocytes from clotted versus unclotted whole blood, including upregulation of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in LPS-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent, but fibrin-dependent manner. Fibrinolysis initiated shortly after plasma clot formation (i.e., 1-2 hours) reduced the synthesis of IL-8 and MCP-1, while delayed fibrinolysis was far less effective. Consistent with these
in vitro
models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1.

Conclusions:
These findings demonstrate that clots are potent inducers of monocyte gene expression, and that timely fibrinolysis attenuates inflammatory responses. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically-proven efficacy of fibrinolytic drug treatment within hours of stroke onset.
Abstract 356: Adhesion of
Staphylococcus Epidermidis
to Fibrinogen Alters Clot Formation Kinetics and Ultimate Stiffness
Authors: Vitale Carolyn , Ma Tianhui , Solomon Michael J , VanEpps J. Scott
Published: July 2021

Bacterial infection is known to increase the risk for thromboembolism. The mechanism underlying this correlation remains largely unknown. We recently showed that the common pathogen
Staphylococcus epidermidis
retards clot formation, increases clot elasticity and generates a heterogeneous clot structure that remodels over time. Here, we elucidate the mechanism of this process by evaluating the capacity for
S. epidermidis
to bind to fibrinogen as a function of its growth phase. We hypothesized that the effect of
S. epidermidis
on a fibrin clot is related to its propensity toward biofilm formation. Therefore, stationary phase (biofilm-like)
S. epidermidis
will have a more robust effect on clot kinetics and elasticity than exponential phase (planktonic). Furthermore, this difference is mediated by increased adhesion to fibrinogen. Rheometry was used to evaluate the formation and resultant elasticity of fibrin clots with exponential or stationary phase
S. epidermidis
. A functional
in vitro
model was developed to evaluate adhesion of
S. epidermidis
to a fibrinogen coated surface in a continuously flowing environment. Fluorescent labeled exponential and stationary phase
S. epidermidis
were visualized flowing through a parallel plate microfluidic chamber past immobilized fibrinogen. Images were obtained every 3 seconds for 30 min. Bacterial deposition rate and mean adhesion time were quantified by automated image analysis. A paired Student’s t-test was used for statistical analysis. Stationary phase
S. epidermidis
retards clot formation and increases resultant elasticity while exponential phase only slightly reduces elasticity. The bacterial deposition rate onto fibrinogen was significantly (p=0.03) greater for stationary phase (1741 ± 1513 cells/cm
2
· sec
-1
) vs exponential phase (676 ± 270 cells/cm
2
· sec
-1
). The average adhesion time however was similar for exponential and stationary phase cells. Coagulation proteins can provide a framework for bacterial adhesion, biofilm formation and infection. In turn infected thrombi with (biofilm-like) bacteria are stiffer which correlates to more frequent bacterial binding to fibrinogen. This provides a potential molecular mechanism for infection mediated thromboembolic events.