bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 110: B2 Cells Suppress Abdominal Aortic Aneurysms and Increase Peripheral Regulatory T Cells
Authors: Meher Akshaya K , Johnston William F , Lu Guanyi , Pope Nicolas , Bhamidipati Castigliano M , Harmon Daniel B , Su Gang , Zhao Yunge , McNamara Coleen A , Upchurch Gilbert R , Ailawadi Gorav
Published: March 2022

Objective:
Recent reports have documented abdominal aortic aneurysm (AA) rupture in patients receiving B cell depletion therapy. This highlights the importance of understanding the role of B cells (B1 and B2 subtypes) in the development of AA. We hypothesized that B2 cells are abundant in AA and aggravate experimental aneurysm formation.

Methods:
AAs were induced in WT (C57BL/6) mice by elastase perfusion of the abdominal aorta and harvested at 14 days. Aortic B1 and B2 cells were quantified, and, intracellular cytokine synthesis was determined using flow cytometry. Next, AAs were induced in B cell deficient (muMT) mice using either elastase perfusion or topical elastase application to determine the impact of loss of B cells in AA. Finally, adoptive transfer experiments were performed on muMT mice by tail vein injection of 25x10^6 isolated splenic B2 cells or saline. Regulatory T cell (Treg) population was identified as CD4+ T cell expressing Foxp3 (forkhead box P3).

Results:
B cells appeared in abdominal aortas at day 7 after elastase perfusion in WT mice and persisted at least till day 21. Compared to saline perfused or normal aortas, elastase perfused aortas had greater infiltration of mononuclear cells including B cells (3631±578 in elastase vs 1476±524 in saline, p=0.02) and T cells. B2 cells constituted 93% of total B cells in AA. Furthermore, B cells isolated from elastase and saline perfused aortas displayed high, but similar levels of intracellular IL-6 and IL-10 expression. Interestingly, in both experimental models of AA, muMT mice were prone to AA formation similar to WT mice as evidenced by elastin degradation or infiltration of T cells, neutrophils and macrophages. Contradicting our hypothesis, adoptive transfer of B2 cells suppressed AA formation (102.0±7.3% vs 75.2±5.5%, p=0.01) with concomitant increase in Treg cells in spleen (0.24±0.03% vs 0.92± 0.23%, p=0.02) and aorta (0.37±0.09% vs 1.09±0.38%, p=0.08), and decrease in aortic infiltration of mononuclear cells.

Conclusions:
Our data suggest that B2 cells constitute the largest population of B cells in experimental AA and participate in protecting aorta from aneurysm formation at least in part by increasing the Treg cell population.
Abstract 130: Immediate Stabilization of Subarachnoid Hemorrhage by Intranasal Delivery of Novel Therapeutics
Authors: Zeng Ning , Peng Tao , McPherson David , Huang Shao-ling
Published: March 2022

Background:
Stroke treatment is a challenge because of the short time window. Intranasal therapeutics delivery is a promising administration options for early delivery of brain stabilization agent as both brain and nasal compartments are interconnected via the olfactory/trigeminal structure and the peripheral circulation. Due to the stabilization effect of nitric oxide (NO) on endothelium, vascular smooth muscle, polymorphonuclear neutrophils (PMNs) in acute cellular and tissue injury, NO releasing agents has been used for emergent cardiovascular conditions such as acute cardiac ischemia and stroke. Here we investigated an intranasal carrier/NO therapeutic delivery strategy to the brain for acute treatment in hemorrhage stroke.

Methods:
NO/argon (1:9) in caged molecular, cyclodextrin, in liposomal formulation was created to carry NO. A total 200 ul (50 ul per 2 minutes х 4 times) of the NO liposomal formulation or a liposomal carrier control was administrated intranasally at 30 minutes after subarachnoid hemorrhage (SAH). Distribution of NO in brain was imaged with DAF-FM. Hemorrhagic volume was quantified by spectrophotometric hemoglobin assay two hours after SAH.

Results:
NO distribution was noted in normal brain tissue (Fig.1A). SAH rats had reduced NO distribution due to hemoglobin scavenging of NO (Fig.1B). Intranasal NO liposomal delivery enhanced NO distribution especially in the cerebral microvascular in SAH brain (Fig. 1C). Effective intranasal NO liposomal delivery resulted in decreased hemorrhagic volume (115 ± 22μl) vs SAH without treatment control (344 ± 63μl, p=0.014) as well as SAH with intranasal liposomes control (298 ± 114μl, p=0.02) (Fig. 1D).

Conclusions:
The Intranasal delivery route of liposomal carriers efficiently delivers therapeutics into the brain. These carriers have controlled release properties in addition to their therapeutic payload effects. The present method has great potential for immediate delivery in stroke.
Abstract 131: Mitogen-Activated Protein Kinase Kinase Activation is Necessary for Macrophage Accumulation, Compensatory Growth of Blood Vessels and Muscle Regeneration Following Femoral Artery Ligation
Authors: Andersen Nicholas J , Boguslawski Elissa A , Naidu Agni , Kits Kara , Bromberg-White Jenn L , Holly Brittany , Kuk Cynthia Y , Winn Mary E , Chang Ting-Tung , Chambers Christopher M , Duesbery Nicholas S
Published: March 2022

Lower limb peripheral vascular disease (PVD) results from the occlusion of arteries leading to reduced blood flow and limb ischemia. Compensatory growth of blood vessels may be sufficient to overcome limb ischemia but for some patients therapeutic intervention, even limb amputation, is required. To gain insight into the mechanisms regulating compensatory growth of blood vessels we used a mouse model of hindlimb ischemia. The ischemic lesions were confirmed and compensatory arteriogenesis was evaluated by high frequency power Doppler ultrasound. In this model, compensatory growth occurs mainly through arteriogenesis. While signaling by mitogen activated protein kinase kinases 1 and 2 (MEK 1 and 2) is required for developmental and tumor angiogenesis, it is unknown whether MEK1/2 activity drives other physiologic vascular growth such as arteriogenesis. We hypothesized that MEK 1/2 activity is necessary for hindlimb re-vascularization following femoral artery ligation. To test this we ligated the femoral artery in mice and then treated them with PD0325901, an allosteric MEK1/2 inhibitor. Following femoral artery ligation active MEK signaling was detected in spindle-shaped cells located within regions of intense angiogenesis and myocyte proliferation. MEK inhibition with PD0325901 reversibly blocked neovascularization, muscle regeneration, and caused extensive coagulative necropathy. MEK inhibition not only prevented arteriogenesis in ischemic limbs but also caused a reduction in arterial diameter. Finally, MEK inhibition prevented accumulation of CD68+ cells in ischemic tissues and skewed systemic cytokine expression towards a persistent, pro-inflammatory phenotype. Our investigation provides pharmacologic evidence demonstrating an essential role for MEK signaling in angiogenesis and arteriogenesis in response to hind limb ischemia. Further, we observed activation of MEK signaling is required for accumulation of macrophages in ischemic tissues and prevents down-regulation of systemic pro-inflammatory cytokines. Lastly, since the effects of MEK inhibition on reperfusion are reversible, we can use MEK inhibitors to create a mouse model of chronic limb ischemia that mimics aspects of PVD.
Abstract 132: Risk Factors for Lower-Extremity Claudication in South Asians
Authors: Damrauer Scott M , Zhao Wei , Fairman Ronald M , Rader Daniel J , Saleheen Danish ,   the PROMIS Investigators
Published: March 2022

South Asians have a higher incidence of CAD and diabetes than people of Western descent. Despite this, the rates of peripheral arterial disease (PAD) have been reported to be lower and the risk factors have not been well characterized. To better understand the determinants of PAD in this population we examined participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), a multi-center case control study of myocardial infarction in Pakistan, for clinical evidence of claudication. 724 (4.8%) of the 15,570 individuals enrolled in PROMIS had symptoms consistent with claudication. These subjects had an average (SD) age of 56 (9.7) years and 536 (74%) were male. Multivariate logistic regression identified the following independent risk factors: age (OR 1.03, 95%, CI 1.02-1.04), female gender (OR 1.7, 95% CI 1.2 - 2.2), ethnicity (OR 1.4 - 4.6 with Punjab as reference), MI (OR 2.2, 95% CI 1.8 - 2.8), prior stroke (OR 2.6, 95% CI 0.9 - 6.2), diabetes (OR 1.3, 95% CI 1.0 - 1.7), family history of hypertension (OR 1.3, 95% CI 1.0 - 1.7), consumption of fried foods (OR 1.8, 95% CI 1.3 - 2.3), sedentary occupation (OR 1.6, 95% CI 1.3 - 2.0), overall sedentary lifestyle (OR 2.5, 1.9 -3.4), being married (OR 1.6, 95% CI 1.0 - 2.8), having a consanguineous spouse (OR 1.3, 95% CI 1.0 - 1.6), minimal formal education (OR 1.9, 95% CI 1.5 - 2.3), and low income (OR 1.2, CI 1.0 - 1.7). The use of ghee (clarified butter) as compared to oil or lard for cooking was protective (OR 0.6, 95% CI 0.5 - 0.8). Although smoking (OR 1.4, 95% CI 1.2 - 1.6) and hypertension (OR 1.3, 95% CI 1.1 - 1.6) were significant risk factors when controlling for age, gender, and ethnicity, they were not independently associated with claudication after adjusting for the other above risk factors (p=0.29 and p=0.28, respectively). These findings suggest that although there is a difference in the prevalence of PAD between South Asian and Western populations, the populations share many of the same risk factors. The association of claudication with spousal consanguinity, as well as female gender and ethnicity, suggest a significant genetic influence that may differ from Western populations and merits further investigation.
Abstract 142: Transcriptome-wide Expression Quantitative Trait Loci Mapping for Atherosclerosis Susceptibility Reveals Surf4 as Novel Candidate Gene
Authors: Northoff Bernd H , Sass Kristina , Nicolaou Alexandros , Teupser Daniel , Holdt Lesca
Published: March 2022

Previous work in an F2 intercross between atherosclerosis-susceptible C57BL/6 and atherosclerosis-resistant BALB/cByJ (BALB) mice on the LDL-receptor deficient background revealed a quantitative trait locus (QTL) for atherosclerotic lesion size at rs33142586 on chromosome (Chr) 2 in female mice (n=183) (Burkhardt et al., Arterioscler Thromb Vasc Biol; 31:775-84, 2011). The aim of the present study was to identify genetic variants responsible for differences in atherosclerosis-susceptibility between the two strains. We hypothesized that the causal genes could be identified by transcriptome-wide expression analyses followed by functional validation.

25,697 transcripts were analyzed in aortas (n=165) and livers (n=176) of F2 mice using Illumina Ref8 expression arrays. Expression levels were correlated with single nucleotide polymorphism (n=31) on Chr2 by expression QTL (eQTL) mapping. Genes co-segregating with the atherosclerosis QTL were identified as potential candidate genes. Results were validated by quantitative real-time PCR (qRT-PCR) in livers (n=176) and bone marrow-derived macrophages (BMDM, n=184) of F2 mice. Expression patterns of candidate genes were examined in different tissues of F0 mice, followed by functional studies.

eQTL mapping identified
Surf4
as the only
cis
-regulated gene co-segregating with the Chr2 QTL in aortas (LOD
array
=17.6), livers (LOD
array
=11.5, LOD
qRT-PCR
=23.2) and BMDM (LOD
qRT-PCR
=15.7).
Surf4
mRNA was significantly increased in F2 mice carrying the BALB allele at rs33142586 and BALB F0 mice. Increased
Surf4
expression was associated with reduced atherosclerosis lesion size in F2 mice. Immunohistochemical staining revealed expression of
Surf4
in vascular endothelial cells of the aorta. RNAi of
Surf4
lead to increased staurosporine- and cycloheximide-induced apoptosis and decreased proliferation compared to control siRNA in RAW 264.7 cells, which are two key mechanisms of atherosclerosis.

In conclusion, we identified
Surf4
as a novel candidate gene of atherosclerosis susceptibility co-segregating a QTL for atherosclerosis on mouse Chr2. In current work, its functional role in atherogenesis is being further investigated in
in-vitro
experiments.
Abstract 145: Palmitic Acid Stimulates a Delayed but Extremely High Expression of IL-6 and Also Enhances LPS Signaling via Sphingolipid Metabolism in Human Aortic Endothelial Cells
Authors: Li Yanchun , Lu Zhongyang , Lopes-Virella Maria F , Huang Yan
Published: March 2022

It is known that both saturated fatty acids (SFAs) and lipopolysaccharide (LPS) boost host chronic inflammatory response that contributes to inflammation-related diseases such as diabetes and atherosclerosis. However, it remains unclear how SFAs interact with LPS to promote inflammation. In this study, we compared the effects of LPS, palmitic acid (PA), a most abundant SFA, and the combination of LPS and PA on the expression of interleukin (IL)-6, a key pro-inflammatory cytokine, in human aortic endothelial cells (HAECs). Results showed that while LPS induced a quick but brief IL-6 mRNA expression that peaked at 12 h, PA stimulated a delayed but extremely high expression of IL-6 mRNA (22-fold of that stimulated by LPS) at 36 h (IL-6 mRNA/house-keeping gene mRNA ratios: control-0.21; LPS-0.4; PA-8.68). Results also showed that the combination of LPS and PA further increased IL-6 mRNA at 36 h (IL-6 mRNA/house-keeping gene mRNA ratios: PA-8.68; LPS plus PA-14.23). Consistently, quantification of IL-6 protein in culture medium showed that PA stimulated more IL-6 secretion than LPS and PA in the combination of LPS led to a much greater IL-6 secretion at 36 h (control-269; PA-2485; LPS-1258; LPS plus PA-5811 pg/ml). Since PA is known to affect ceramide biosynthesis, we determine if sphingolipid metabolism is involved in IL-6 upregulation by LPS, PA or LPS plus PA. Results showed that PA significantly increased sphingomyelin (SM) hydrolysis and PA in combination with LPS further increased it (cellular SM levels: control-18.5; LPS-16.5; PA-13.6; LPS plus PA-10.7 pmol/ng phosphate). Increased SM hydrolysis was associated with an increase in ceramide, a bioactive sphingolipid known to stimulate inflammatory cytokines. Moreover, using pharmacological inhibitor and RNA interfering, we found that neutral sphingomyelinase (nSMase), but not acid SMase, was involved in the SM hydrolysis. Finally, we found that inhibition of SM hydrolysis by nSMase inhibitor or nSMase siRNA markedly inhibited IL-6 expression stimulated by LPS, PA or LPS plus PA. In conclusion, this study demonstrated that PA not only stimulated a delayed but extremely high expression of IL-6, but also enhanced the effect of LPS in HAEC, and sphingolipid metabolism played a crucial role in the PA action.
Abstract 150: Trastuzumab-Associated Inflammatory Responses in Healthy Volunteers: Possible Insights in Immunogenicity of Monoclonal Antibodies
Authors: Reijers Joannes A , Moerland Matthijs , Burggraaf Jacobus
Published: March 2022

Many monoclonal antibodies are associated with an inflammatory reaction. For example, trastuzumab induces such a response in 40-60% of treated patients, most often after the first administration. Apart from causing discomfort for the patient, these responses may increase the risk of antibody formation against the therapeutic agent. Not much is known on the pathways involved, however, comparing clinical findings and results of in vitro cytokine release between subjects who did and did not develop an inflammatory reaction may offer guidance for further investigations.

A cohort of 54 healthy male volunteers randomly received either placebo (n=8) or 6 mg/kg trastuzumab (n=46) intravenously in 90 minutes. Adverse events were captured and regular safety assessments were performed, consisting of vital signs and routine clinical laboratory tests. In addition, whole blood from a selection of volunteers with clinical signs of an inflammatory reaction as well as whole blood from trastuzumab-naïve healthy volunteers was incubated in vitro with trastuzumab.

Fever was observed in 9 (19.6%) volunteers receiving trastuzumab, occurring in combination with other influenza-like symptoms in 4. Seven subjects reached a temperature of ≥38.5°C, and four ≥39.0°C, with a maximum of 39.4°C. One volunteer developed influenza-like illness without fever. The mean latency between the onset of complaints and start of infusion was 4.7 h (SD 2.6). The fever was accompanied by a mean increase in leucocytes of 54% (SD 31), predominantly neutrophils (increase 128%, SD 60), at 24 h post administration. Although differences in baseline characteristics were not observed, a rise in neutrophils (mean increase at 24 h 94%, SD 55) and body temperature (mean peak value 37.3°C, SD 0.4) was also noted in the 37 subjects who did not develop fever. None of the placebo treated volunteers displayed this phenomenon.

These results suggest that 6 mg/kg trastuzumab is capable of inducing an inflammatory response, although the magnitude and thus the clinical manifestations differ between subjects. Data on the correlation between in vitro cytokine release, which might serve as a platform for immunogenicity research, and the observed clinical response will be presented.
Abstract 163: Cell-Surface Protein Disulfide Isomerase Shapes Vascular Architecture to Counteract Inward Remodeling
Authors: Araujo Haniel A , Tanaka Leonardo Y , Hironaka Gustavo K , Takimura Celso K , Rodriguez Andres I , Gutierrez Paulo S , Laurindo Francisco R
Published: March 2022

BACKGROUND:
Vascular remodeling orchestrates a complex network of signaling pathways responsible for pathological changes in many vascular diseases such as atherosclerosis. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI (ecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI).

Methods and Results:
After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and ecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis). PDI knockdown also upregulated proliferation marker PCNA and decreased differentiation marker calponin-C. EcPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. EcPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and induction of thicker collagen fibrils. Collagen type I and III were decreased after ecPDI inhibition in arteries 14 days AI. Furthermore, ecPDI inhibition prevents injury-increased hydrogen peroxide generation and decreases arterial nitrate (NO3-) levels. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. There was decreased PDI expression in media and intima from plaques exhibiting inward remodeling and, conversely, enhanced PDI expression in media of plaques exhibiting outward remodeling.

CONCLUSIONS:
Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect. Such findings suggest an important role for PDI in lumen maintenance during vascular remodeling.
Abstract 164: The Role of IEX-1 in Venous Stenosis Formation Associated With Arteriovenous Fistula Used for Hemodialysis
Authors: Brahmbhatt Akshaar , Yang Binxia , Nieves-Torres Evelyn , McCall Deborah , Mukhopadhyay Debabrata , Kumar Rajiv , Misra Sanjay
Published: March 2022

Purpose:
The majority of patients with end stage renal disease who require hemodialysis use a arteriovenous fistula (AVF) for their vascular access. However, only 40% of AVFs are clinically usable after 1 year. Arteriovenous fistulas fail because of venous neointimal hyperplasia (VNH) that develops in the proximal outflow vein. The immediate early gene, IEX-1 (IER-3), is increased in response to shear stress and hypoxia and both factors contribute to VNH. IEX-1 expression was increased in stenotic venous stenoses removed from patients with hemodialysis vascular access compared to controls. We hypothesized that reducing the expression of IEX-1 using a knockout mouse model with chronic kidney disease (CKD) and AVFs would have decreased VNH compared to wild type (WT) mice.

Methods:
CKD was induced by performing a nephrectomy. Four weeks later, an AVF was placed to connect the carotid artery to ipsilateral jugular vein. The mice were sacrificed 3 and 28 days after fistula creation and outflow veins were harvested for gene expression and histomorphometric analyses.

Results:
By day 3, the average gene expression of vascular endothelial growth factor-A (VEGF-A) was significantly lower in the IEX-1 KO vs. WT mice (average reduction: 40%, P<0.05). At day 28, the average lumen vessel area was increased significantly (average increase: 133%, P<0.05, IEX-1 KO vs. WT, respectively) while the average area of the neointima was significantly decreased (average reduction: 20%, IEX-1 KO vs. WT, respectively). Accompanied with this, there was a significant decrease in α-SMA staining and Ki-67 proliferation while the average TUNEL staining was significantly increased (all P<0.05, IEX-1 KO vs. WT). Finally, there was a significant decrease in the average staining intensity for VEGF-A and matrix metalloproteinase-9 (MMP-9) in the KO vs. WT mice (both, P<0.05).

Conclusion:
A reduction in IEX-1 gene expression results in positive vascular remodeling accompanied with a reduction in proliferation and α-SMA density mediated through a VEGF-A/MMP-9 pathway. Taken collectively, these findings indicate that reducing IEX-1 expression is protective for VNH formation and provide a rationale for use of anti-IEX-1 therapies aimed at prolonging AVF function.
Abstract 18: Circulating Extracellular DNA Mediates Vaso-Occlusions in Sickle Cell Disease
Authors: Le Jeune Sylvain , Bonnin Philippe , Charue Dominique , Arlet Jean-Benoît , Lionnet Francois , Tedgui Alain , Tharaux Pierre-Louis , Boulanger Chantal , Blanc-Brude Olivier
Published: March 2022

Rationale:
It was recently reported that circulating extracellular DNA (cirDNA) is increased in sickle cell disease (SCD). CirDNA apears to be associated to intravascular hemolysis and cell-free heme in plasma. But nothing is known of the physiopathological impact of cirDNA in SCD. Here, we aimed to define the role of cirDNA and hypothesized that it may favor red blood cell (RBC) aggregation, vaso-occlusive crises (VOC) and ischemic events.

Material and Methods:
A blood collection from SCD patients and matched healthy volunteers was built. RBCs and neutrophils were separated from plasma by centrifugation. We cultured human neutrophils and endothelial cells in vitro. We quantified extracellular DNA with a fluorescent intercalant probe. We also used our model of renal vaso-occlusions induced by infusion of heme-loaded RBC microparticles in SAD transgenic mice.

Results:
In SCD patients at steady state, cirDNA was increased by 300% over health, and a further 100% during VOC. Next, we treated cultured neutrophils and endothelial cells with purified heme. Heme induced neutrophil extracellular traps (NETs) and endothelial apoptosis, with release of genomic DNA in supernatants. We purified neutrophil DNA, added it to whole blood and studied RBC aggregation by laser-assisted optical red blood cell aggregometry. DNA induced RBC aggregation in SCD blood, but not healthy blood. Conversely, DNase-1 added to SCD blood collected during VOC reduced RBC aggregation back to steady state levels. Next, we triggered renal vaso-occlusions in SAD transgenic mice, and cirDNA levels were increased by 200% within 30 minutes. Moreover, one intravenous bolus of DNase-1, administered after inducing the occlusions, accelerated renal reperfusion back to normal levels in under 20 minutes. In contrast, spontaneous reperfusion with vehicle injection took over 90 minutes.

Conclusion:
Our data reveal a novel mechanism of vascular occlusion in SCD: CirDNA can bind RBCs in blood, promote aggregation and small vessel occlusions. CirDNA and NETs may thus bridge intravascular hemolysis, inflammation and ischemic tissue injury in SCD. Interestingly, DNase digestion of plasma DNA and NETs may prove an unexpected but powerful novel pharmaceutical approach to treat vaso-occlusive events.
Abstract 184: Alterations in Tissue-Based Sca-1+ Stem Cell Populations in Diabetic Arteries
Authors: Streams Jill R , Shively Vera P , Vercammen Janet M , Flynn Megan E , Kibbe Melina R
Published: March 2022

Objectives:
The stem cell antigen-1 (Sca-1) progenitor cell is a pluripotent stem cell that is implicated in homeostasis of the vascular wall, including regulation of the response to injury. Diabetic mice have a decreased Sca-1+ bone marrow population, but it is not known how the diabetic state affects the permanent tissue-based Sca-1+ population. We hypothesize that diabetic mice have a decreased Sca-1+ stem cell population in the arterial wall compared to wild type mice.

Methods:
Arteries were harvested from male C57BL/6 wild type (WT) and Lepr Db/db diabetic (DB) mice (n=4-6/strain). Diabetic state was confirmed in DB mice by measuring serum insulin and glucose levels. Harvested segments included carotid, thoracic aorta, abdominal aorta, and femoral arteries. Arteries underwent immunofluorescent staining for Sca-1. Staining in the adventitia, media and intima was assessed by blinded grading (scale of 0-4).

Results:
Sca-1+ stem cells were predominantly located in the adventitial layer of the arterial wall in both WT and DB mice (7.2-fold vs. media, 3.5-fold vs. intima). In the carotid artery, DB mice demonstrated 26% less adventitial Sca-1+ staining than WT mice (1.69±0.1 vs. 2.27±0.1, P≤0.001). In the thoracic aorta, DB mice had 28% less adventitial Sca-1+ staining than WT mice (2.06±.0.1 vs. 2.88±0.1, P≤0.001). Notably, no significant difference was detected in adventitial Sca-1+ staining in the abdominal aorta or femoral arteries between the mouse strains. In the media, DB mice showed a trend towards increased Sca-1+ staining; this only reached significance in the abdominal aorta (0.55±0.1 vs. 0.22±0.1, P=0.011). In the intima, there was a trend towards increased Sca-1+ staining in DB mice among all arterial segments, but this did not reach statistical significance.

Conclusions:
Diabetic mice demonstrate a depleted Sca-1+ stem cell population in the adventitia of the carotid artery and thoracic aorta compared to WT counterparts. In contrast, diabetic mice showed an increased Sca-1+ population in the media of the abdominal aorta. This overall decrease in the adventitial Sca-1+ stem cells of diabetic mice and shift in distribution amongst the arterial wall layers may contribute to the altered homeostasis and response to injury in diabetic vessels.
Abstract 195: Characterization of DNA Modules in Regulating VWF Vascular Bed--Specific Expression
Authors: Yuan Lei , Beeler David , Spokes Kate , Janes Lauren , Chan Gary , Regan Erzsebet , Aird William C
Published: March 2022

Expression of von Willebrand Factor (vWF) in the endothelium is regulated by vascular bed-specific pathways. A region of the human vWF gene between -843 and -620 was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to further delineate the responsible DNA sequences. A series of constructs containing 5’-deletions, internal deletions or point mutations of the vWF promoter coupled to LacZ were targeted to the Hprt locus of mice using homologous recombination, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that a three-base-pair fragment within vWF promoter is necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Although the sequence does not conform to established DNA binding sites, it bound to nuclear protein as determined by elecrophoretic mobility shift assay. Mass spectrometry of the DNA-protein complex revealed several potential binding transcription factors. The heart- and skeletal muscle-specific promoter region did not response to shear stress or mechanical stress. Bisulfite sequencing revealed differential methylation of the human vWF promoter in endothelial cells from expressing and non-expressing vascular beds. These differences were altered by the mutation at the short cis-element. Together, these data support a model of modular gene regulation in which distinct DNA sequences in the vWF promoter direct expression in different vascular beds.
Abstract 198: Activation of Human Platelets by Staphylococcus aureus Secreted Protease Staphopain A
Authors: Clarke Simon R
Published: March 2022

Staphylococcus aureus is the leading aetiological agent of infective endocarditis (IE). The pathogen can induce the activation of platelets, resulting in aggregation and thrombus formation, which are regarded as important steps in the pathogenesis of IE. In this study, we demonstrate that staphopain A, a cysteine protease secreted by S. aureus, induces the aggregation of human platelets. The presence of staphopain A in the supernatant of S. aureus confers an ability to activate platelets, in addition to the actions of alpha haemolysin also released by the pathogen. Staphopain A platelet agonist activity was inhibited by addition of the specific inhibitor staphostatin A, implicating its protease activity in the agonism. Concentrations of staphopain A that were insufficient to induce platelet aggregation caused increased platelet binding to collagen. Using specific antagonists, protease-activated receptors 1 and 4 were shown to be responsible for mediating staphopain A induced activation of platelets.
Abstract 20: RhoA GTPase Regulates Reactive Oxygen Species--Mediated Platelet Activation
Authors: Akbar Huzoor , Duan Xin , Saleem Saima , Davis Ashley K , Zheng Yi
Published: March 2022

Rho family GTPases and reactive oxygen species (ROS) are known to be involved in platelet activation. Although Rac1, a Rho member, has been shown to be critical for ROS generation by NADPH oxidase (NOX) and platelet activation, RhoA, the founding member of the Rho family, has not been suspected in ROS generation and associated platelet activation. In this study we investigated the effects of Rhosin, a rationally designed small molecule that specifically binds to RhoA at the site required for its activation by Rho-GEF and thereby prevents activation of RhoA (Chem. Biol. 19, 699-710, 2012) on ROS generation and platelet activation to better understand the role of RhoA in platelet signaling. Addition of Rhosin to washed human platelets two minutes prior to stimulation with U46619, a stable analog of TXA
2
, inhibited (a) activation of RhoA but not of Rac1 or Cdc42, (b) ROS generation, and (c) phosphorylation of p47
phox
, a critical component of the NOX complex. In other experiments, U46619 induced ROS generation in platelets from RhoA
+/+
but not in RhoA
-/-
mice. Treatment of platelets with Rhosin inhibited U46619-induced (a) phosphorylation of myosin light chain (MLC) in the presence or absence of aspirin and apyrase, (b) platelet shape change, filopodia formation and spreading on immobilized fibrinogen, (c) release of P-selectin, secretion of ATP and aggregation, and (d) phosphorylation of ERK and p
38
-MAPK, and Akt. These findings suggest that RhoA induced ROS generation contributes to platelet activation via phosphorylation of p
38
-MAPK, ERK and Akt. Rhosin also inhibited thrombin induced ROS generation, platelet shape change, secretion, aggregation and clot retraction. Washing of platelets after incubation with Rhosin abolished the inhibitory effects of Rhosin on platelet activation. Administration of Rhosin to mice prolonged tail bleeding times. These data suggest that: (a) agonist-RhoA/ROCK mediated phosphorylation of p47
phox
plays a critical role in ROS generation which in turn induces platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) targeting RhoA by rationally designed small molecule inhibitors offers a novel approach for developing safer and more effective antithrombotic agent.
Abstract 202: Inhibition of Central Melanocortin 4 Receptor Signaling Severely Impairs Brown Adipose Tissue Activity and VLDL Metabolism
Authors: Berbée Jimmy F , Kooijman Sander , Boon Mariëtte R , Parlevliet Edwin T , Geerling Janine J , Havekes Louis M , Meurs Illiana , Rensen Patrick C
Published: March 2022

Objectives:
SNPs near the melanocortin 4 receptor (MC4R) gene are associated with cardiovascular diseases (CVD) and deficiency of MC4R is the most common monogenic cause of obesity, a major risk factor for the development of CVD. MC4R-deficient individuals not only show increased food intake but also lower basal energy expenditure. Furthermore, pharmacological inhibition of the central MC4R signaling by SHU9119 in mice increases weight gain independent of food intake. Since brown adipose tissue (BAT) importantly contributes to energy expenditure by combusting high amounts of triglycerides (TG) into heat, the aim of the study was to evaluate the effect of inhibiting MCR4 on VLDL-metabolism and BAT activity in mice.

Methods and results:
Female
APOE*3-Leiden.CETP
transgenic mice were fed a cholesterol-enriched Western-type diet for 4 weeks, matched on body weight and plasma lipids, and infused with either SHU9119 or vehicle for 2 weeks via an osmotic mini-pump into the lateral ventricle while being fed the same diet. SHU9119 increased body fat (+50%) and decreased fat oxidation (-42%). Shu9119 increased plasma VLDL-TG levels (+30%). Whereas hepatic VLDL-TG production rate was not affected, Shu9119 severely impaired clearance of VLDL-TG, explained by reduced uptake of [
3
H]TG by BAT (-25%). In BAT, SHU9119 decreased UCP-1 protein (-60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Since SHU9119 increased food intake (+30%), SHU9119 was also administered in mice that were pair-fed with the vehicle-treated group. Under these conditions, SHU9119 still exhibited these effects, indicating MC4R inhibition impairs BAT activity independent of food intake.

Conclusion:
Inhibition of central MC4R signaling by SHU9119 increases VLDL-TG levels and decreases energy expenditure specifically by impairing BAT function. We anticipate that MC4R regulates adiposity and VLDL metabolism not only by regulating food intake, but also by modulating BAT activity, and that activation of MC4R is a promising strategy to combat obesity and CVD by increasing BAT activity.
Abstract 204: Early Life Predictors of Augmentation Index in Biracial (Black-White) Young Adults: The Bogalusa Heart Study
Authors: Fernández Alonso Camilo , Li Shengxu , Galazka Patrycja , Chen Wei , Srinivasan Sathanur R , Berenson Gerald S
Published: March 2022

Background:
The central pressure augmentation index (AI) has been suggested as a noninvasive measure of pulsatile load, which is a likely a determinant of left ventricular mass and other cardiovascular outcomes. While adult risk factor associations with AI have been documented, information is scant on the childhood predictors of AI in relatively young and healthy adults.

Hypothesis:
childhood systolic blood pressure is associated with adult AI.

Methods:
this aspect was examined in 820 non-institutionalized individuals (65.2% whites, 53.8% females; aged 29.4 - 51.3 years) that participated in the Bogalusa Heart Study and had cardiovascular risk factors information available since childhood. AI was estimated in adulthood through applanation tonometry of the radial artery. The average value of childhood measurements was used as the childhood value, standardized to age, race and sex-specific z-scores. Multivariable linear regression analyses were used to estimate childhood risk factor associations with AI in adulthood.

Results:
Blacks and males had significantly higher levels of AI in adulthood, compared to their counterparts. After controlling for the effect of age, race and sex, systolic blood pressure (SBP) measured in childhood resulted as the only significant predictor or AI in young adults (β= -0.26, p=0.03). No significant associations were observed in other childhood risk factors such as: high density lipoprotein cholesterol, low density lipoprotein cholesterol, body mass index or diastolic blood pressure.

Conclusion:
These findings support the hypothesis that SBP in childhood has an impact on adult pulsatile load (as measured by means of AI). Further, this information stresses the importance of primordial prevention of CV risk factors at an early age, as these are responsible of overt cardiovascular disease and complications in adulthood.
Abstract 210: Hyperglycemia Results in an Exaggerated Response to Ischemia in Human Dermal Fibroblasts Through MyD88 Dependant Toll-Like Receptor 4 Activation
Authors: Portou Mark J , Yu Rebekah , Shi-wen Xu , Abraham David , Hamilton George , Baker Daryll , Tsui Janice
Published: March 2022

Introduction:
Foot ulceration is a multifactorial, frequent and challenging complication of diabetes. There is increasing recognition of diabetes as a pro-inflammatory condition and evidence that toll-like receptor 4 (TLR4) activation is involved in its systemic pathogenesis and the subsequent impairment of wound healing, however the mechanism remains unclear. We aim to study the effect of simulated diabetic-ischaemic conditions on TLR4 function in dermal fibroblasts.

Method:
Human fibroblasts were cultured at physiological glucose concentration (5.5mM) and subsequently exposed to glucose concentrations from 0mM to 25mM for 24 hours. Identical samples were placed within a hypoxic chamber for 8 hours. Cell lysate protein and supernatant were harvested and western blot assays for TLR4, MyD88 and cleaved Caspase 3 were undertaken. Migration was assessed by scratch wound assay. The effects of MyD88 and TRIF inhibitory peptides and TLR4 neutralising-antibody and antagonist on fibroblast migration were assessed in the 25mM glucose groups.

Results:
Hypoxic conditions led to an increase in TLR4 protein expression. This effect was significantly increased (p<0.05) in very high glucose concentrations (25mM), and resulted in increased apoptosis (cleaved caspase3) and IL-6 release. Hypoxic conditions resulted in impaired fibroblast migration, particularly at high glucose concentrations (p<0.05). Inhibition with a MyD88 inhibitory-peptide and a TLR4 neutralising antibody and antagonist ameliorated the effects of high glucose and ischaemia (p<0.05).

Conclusion:
Hypoxia leads to increased TLR4 protein expression and this effect is exaggerated by hyperglycaemia. This results in an increase in cellular apoptosis. The migration of fibroblasts in hypoxia was disproportionately impaired in the very high glucose treatment groups. TLR4 and MyD88 inhibition resulted in significantly improved fibroblast migration under these conditions. We conclude that inflammatory processes mediated via the MyD88-dependent TLR4 pathway results in reduced migration of fibroblasts and may contribute to impaired wound healing, under conditions commonly found in diabetic patients with concomitant ischaemia.
Abstract 214: Effect of Intensive Lipid-Lowering Therapy on Epicardial Adipose Tissue in Patients Undergoing Pulmonary Vein Isolation for Atrial Fibrillation
Authors: Soucek Filip , Covassin Naima , Singh Prachi , Ruzek Lukas , Kara Tomas , Suleiman Mahmoud , Koestler Celeste , Friedman Paul A , Somers Virend K
Published: March 2022

Background:
The epicardial adipose tissue (EAT) has been identified as a sensitive marker of cardiometabolic risk. Recent evidence suggests efficacy of long-term statin therapy in reducing the EAT in patients with coronary artery disease. Whether the volume of EAT changes in patients with atrial fibrillation (AF) treated with a statin for a short time period is currently unknown.

Methods:
A population of AF patients undergoing pulmonary vein isolation was randomized to receive either 80 mg/day of atorvastatin (n=51, 44 males, age 55±10 years) or placebo (n=51, 41 males, age 56±10) for a 3 month period. EAT volume was assessed by cardiac computed tomography at baseline and after 3-month follow-up.

Results:
Patients randomized to statin treatment exhibited significant decreases in EAT volume (baseline vs follow-up: 105.8 ± 52.95 vs 103.05 ± 52.55 cm3, p = 0.05), C-reactive protein (2.97 ± 4.3 mg/L vs 2.03 ± 2.2 mg/L, p < 0.05), total cholesterol (182.5 ± 36.9 vs 129.3 ± 40.6 mg/dL, p < 0.001), and low density lipoprotein-cholesterol (111.88 ± 28 vs 61 ± 32.6 mg/dL, p < 0.001) at follow-up, while the body mass index did not vary (29.42 ± 5.2 vs 29.11 ± 4.8 kg/m2, p = 0.28). No changes were seen in the placebo group.

Conclusions:
Short-term high-dose statin therapy significantly reduced the volume of EAT in AF patients. EAT volume modulation might be an important mechanism by which statins may influence cardiovascular risk.
Abstract 216: PCSK9 Loss-of-Function Mutation Q152H is Associated with Reduced Dietary Fat Clearance and Insulin Resistance in Humans
Authors: Wassef Hanny , Bissonnette Simon , Lamantia Valerie , Cyr Yannick , Chrétien Michel , Faraj May
Published: March 2022

Introduction:
PCSK9 regulates plasma LDL-C concentrations. Loss-of-function (LOF) mutations are associated with cardiovascular protection via increased clearance of LDL particles. Work from our and other labs has shown that increased cellular uptake of LDL is proapoptotic for pancreatic β-cells and impairs adipose tissue function. Moreover, obese women with high concentration of LDL (ie plasma apoB) have lower insulin sensitivity and delayed dietary fat clearance. We recently identified a novel PCSK9 LOF mutation, Q152H, causing a 79% reduction of plasma PCSK9 and 48% reduction of plasma LDL-C. The impact of this mutation on the metabolic profile remains unexplored.

Hypothesis:
We hypothesize that despite a cardioprotective profile, carriers of Q152H have delayed dietary TG clearance and impaired insulin sensitivity similar to subjects with high apoB.

Methods/Results:
The heterozygous Q152H proband woman was compared to 2 groups of obese, age, sex and BMI-matched controls with low (n=6) and high (n=7) apoB. Fasting apoB, PCSK9, total cholesterol and LDL-C concentrations were lower in the proband (0.84g/l,126ng/ml, 4.22mM and 2.11mM, respectively) compared to low apoB (0.75 ± 0.05 g/l, 267 ± 18ng/ml, 5.1 ± 0.2mM and 2.8 ± 0.3nM, respectively) and high apoB (1.25 ± 0.09 g/l, 412 ± 79ng/ml, 6.9 ± 0.4mM and 4.3 ± 0.3nM, respectively). Women with high apoB had delayed dietary TG clearance over 6 hours as compared to women with low apoB (incremental TG AUC: 6.75 ± 1.65 vs 4.06 ± 0.77 mM/6h, p=NS). Incremental AUC TG in the proband was 106% greater than that of women with high apoB and 243% greater than that of low apoB. Botnia clamp was also conducted to measure insulin secretion and sensitivity. There was no difference in insulin secretion between the proband and the control groups measured via an intravenous glucose tolerance test. However, insulin sensitivity, as measured by glucose infusion rates (GIR) during a hyperinsulinemia clamp, was 7.5mg/kg/min in the proband, 32% lower than women with high apoB (11.1 ± 1.1 mg/kg/min) and 48% lower than women with low apoB (14.3 ± 0.9 mg/kg/min).

Conclusion:
PCSK9 deficiency favours a cardioprotective profile; however increased clearance of LDL by peripheral tissue may induce peripheral insulin resistance in obese subjects.
Abstract 22: Direct Interaction of Kindlin-3 with Integrins in Platelets and Neutrophils is Essential for Supporting Arterial and Venous Thrombosis in Mice
Authors: Xu Zhen , Gao Juan , Chen Xue , Zhi Huiying , Liu Junling , White Gilbert C , Plow Edward E F , Ma Yan-Qing
Published: March 2022

Kindlin-3 is a critical supporter of integrin activation in hematopoietic cells. Lack of expression of kindlin-3 protein in patients can cause impaired platelet aggregation and leukocyte adhesion, leading to severe bleeding and recurrent infections. Although kindlin-3 has been categorized as one of the integrin binding partners, the functional significance of the direct interaction of kindlin-3 with integrins in platelets and leukocytes has not been established. In this study, we generated a strain of Kindlin-3 knock-in (K3KI) mice that carry a specific mutation in the Kindlin-3 gene, therefore introducing an integrin interaction defective substitution (Q
597
W
598
/AA) in kindlin-3 protein. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbβ3 activation, suppressed platelet spreading and aggregation, prolonged tail bleeding time, absence of platelet-mediated clot retraction and resistance to
in vitro
thrombus formation and
in vivo
arterial thrombosis. In addition, β
2
-integrin mediated adhesion of neutrophils from K3KI mice was dramatically inhibited and, as a consequence, deep vein thrombus formation in a flow-restricted inferior vena cava was significantly suppressed in K3KI mice. Taken together, these observations demonstrate that the direct binding of kindlin-3 to integrin in hematopoietic cells is required for supporting integrin function in platelets and leukocytes, and significantly contributes to both arterial and venous thrombus formation. Our data also suggests that the direct crosstalk between kindlin-3 and integrin represents a potential therapeutic target for developing novel anti-thrombotic strategies.
Abstract 223: The Structural Organization of Apolipoprotein A-I in Reconstituted High-Density Lipoprotein Discs Using Stable Isotope-Assisted Cross-Linking
Authors: Melchior John , Morris Jamie , Davidson W S
Published: March 2022

X-ray crystallography studies of lipid-free apolipoprotein A-I (apoA-I) and related apoA-IV have shown that the proteins form a reciprocating dimer which could be directly related to the structural organization of lipid-bound apoA-I. Previous work has demonstrated the success of chemical cross-linking combined with mass spectrometry to derive distance constraints for molecular modeling of apoA-I in reconstituted high density lipoprotein (HDL) particles. However, these studies have been limited by i) the relative paucity of distance constraint data, and ii) ambiguities in determining the molecular span of the cross-links. Here, we have addressed the first issue by using an affinity-tagged cross-linker to purify cross-linked peptides away from non-modified peptides prior to sampling with mass spectrometry. This essentially boosts the signal from cross-linked peptides of interest that would have otherwise been suppressed by detection of non-modified peptides. Using this method, we identified >25 cross-linked peptides; the most reported to date for these particles. To address the issue of molecular span, we used a stable isotopic-labeling approach in which wild-type human recombinant apoA-I (
14
N) was mixed 1:1 with an isotopically labeled (
15
N) apoA-I. Analysis of the isotopic patterns of the resulting mass spectra allowed the unambiguous determination of the molecular span of the cross-linked peptides; i.e. whether the links were within the same molecule of apoA-I (intramolecular) or between the dimers of apoA-I (intermolecular). This information will be used to derive a detailed novel model of lipid-bound apoA-I in reconstituted HDL, an important step in our overall understanding of how native HDL species are organized and how they interact with plasma remodeling factors and cell surface proteins.