bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 151: Thrombosis Prone Plaques Exhibit Inflammation Prior to Rupture: Intravascular Detection via Macrophage-targeted Optical Imaging
Authors: Stein Ashley F , Hara Tetsuya , McCarthy Jason R , Mauskapf Adam , Ntziachristos Vasilis , Jaffer Farouc A 
Published: March 2022

Introduction
Inflammation modulates atherogenesis and the destabilization of plaques. However, the role of macrophages in plaque rupture is incompletely understood. Here we investigated the in vivo spatial distribution of plaque macrophages prior to triggered plaque rupture, using intravascular molecular imaging and a near-infrared fluorescence (NIRF) macrophage targeted agent, CLIO-CyAm7. We hypothesized that CLIO-CyAm7 would illuminate macrophages in vivo and preferentially localize to plaques that are vulnerable to plaque rupture.

Methods
Atherosclerosis was induced in rabbits (n=14) using a 12-week hyperlipidemic diet with alternating 1% high cholesterol and normal chow, concomitant with aortic balloon injury at 2 weeks. Rabbits were injected with 2.5mg/kg (n=8) or 5mg/kg (n=6) of CLIO-CyAm7 at 24 or 48 hours, respectively, prior to thrombosis triggering using Russell’s Viper Venom and histamine over 48 hours. The 2.5mg/kg subgroup was imaged in vivo using NIRF imaging and intravascular ultrasound (IVUS), immediately prior to thrombosis triggering. After sacrifice, ex vivo imaging, fluorescence microscopy (FM), Carstairs and Ram-11 immunostaining were performed.

Results

FM identified significantly higher CLIO-CyAm7 signal in areas of atherosclerosis compared to normal segments of the aorta (2.5mg/kg and 5.0mg/kg, p=0.03 and p=0.03). This signal was primarily localized at the intimal-luminal border of atheroma, with some penetration in the media and adventitia. CLIO-CyAm7 colocalized with a subset of superficial macrophages. Carstairs staining identified fibrin-rich thrombi, indicating plaque rupture in 6 of 14 rabbits (43%). There was increased CLIO-CyAm7 in areas underlying thrombus and particularly at the shoulder of ruptured plaques. Plaque macrophages were detectable via
in vivo
intravascular NIRF imaging. Areas of atherosclerosis, determined by IVUS, showed significantly higher NIRF peak signal-to-noise ratio than normal segments of the aorta (40.2±16.9 and 5.9±1.9, p=0.02).

Conclusion
Macrophages are consistently present in plaques that undergo triggered rupture, and can be detected via CLIO-CyAm7 intravascular NIRF molecular imaging. In vivo macrophage imaging may identify thrombosis-prone plaques.
Abstract 16: Low Doses of Ionizing Radiation Promote Therapeutic Revascularization in Ischemic Tissues
Authors: Ministro Augusto , Nunes Raquel J , Oliveira Paula , Rocha André , Grillo Isabel , Dinis da Gama A. , Constantino Susana 
Published: March 2022

Lower limb ischemia is a major health problem. Because of the absence of effective treatment in the advanced stages of the disease, amputation is undertaken, even though it is associated to morbidity and mortality. Therefore, novel therapeutic approaches are necessary, aiming at the formation of stable, well-organized and functional blood vessels in ischemic tissues.
Recently, we found that low doses of ionizing radiation (IR) promote angiogenesis and we assessed the hypothesis that they may be therapeutically useful in vascular occlusive disease.

By using a mouse model of hind limb ischemia we quantified the blood flow expressed as a ratio of ischemic to non-ischemic limb perfusion in irradiated and unirradiated mice at different times and our results suggest that, at 15 days post-ischemia, low-dose IR significantly increases perfusion recovery (p=0.00075; n=6 mice per group). Moreover, a statistically significant increase in the capillary density was noted in ischemic irradiated muscles when compared to ischemic unirradiated ones (p=0.00009; n=6 mice per group). On the contrary, no difference was observed when comparing non-ischemic muscles irradiated or not. These results are representative of at least four independent experiments. From an
in vitro
microarray data obtained in our lab, we selected the genes whose expression is significantly altered by low doses of IR and that represent the best candidates for a pro-angiogenic response. Their expression was evaluated in endothelial cells isolated from the gastrocnemius muscles from irradiated and unirradiated mice. A laser capture microdissection microscope was used followed by RNA extraction and cDNA synthesis. Several pro-angiogenic genes were preamplified before quantitative RT-PCR analyses. Our results suggest that low-dose IR induces the expression of VERGR1, VEGFR2, FGF2, TGFB2 and ANG2 in endothelial cells isolated from ischemic muscles.

In conclusion, our data have the potential to propose a new strategy for neovascularization using low doses of IR. The use of a hind limb ischemia model in larger animals will be fundamental to firmly establish the benefits and exclude any harmful effects, before considering low-dose IR as a valid tool to be used in contemporary human medicine.
Abstract 162: Exposure of Human Macrophages to Tobacco Smoke Induces Hmgb1 Release on Microvesicles That Cause Monocyte Recruitment and Impairment of Insulin Signaling in Adipocytes
Authors: Chen Yan , Li Guangping , Liu Yanxia , Williams Kevin , Liu Ming-Lin 
Published: March 2022

Background_
Smokers show increased prevalence of insulin resistance and type 2 diabetes. The underlying mechanisms, however, remain poorly understood. We recently implicated a role for HMGB1, a damage-associated molecular pattern previously shown to be released as a soluble molecule. We reported that exposure of human macrophages to tobacco smoke extract (TSE) provokes the release of HMGB1 in two forms: as a soluble molecule and on membrane microvesicles (MV). The HMGB1 on TSE-induced MVs mediates crucial crosstalk with adipocytes, e.g., these particles stimulate MCP1 secretion by adipocytes and the recruitment of monocytes.

Aims_
To explore the mechanisms by which HMGB1 is released on MVs, and the harmful effects of the TSE-MVs on macrophage-adipocyte crosstalk.

Methods/Results_
To examine how HMGB1 is released on MVs, we found that exposure of human macrophages to TSE upregulated HMGB1 expression, induced its gradually translocation from the nucleus to the cytosol and plasma membrane, in a time-dependent manner, and then the release of HMGB1 both as a soluble molecule and on TSE-MVs. Inhibition of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), by using CRM1 inhibitor III, attenuated the translocation of HMGB1 from the nucleus and its export on microvesicles. To examine biological effects, we found that treatment of adipocytes with TSE-MVs inhibited insulin-induced phosphorylation of IRS1 (Y612) and Akt (S473). Co-incubation of adipocytes with TSE-MVs and human monocytes produced more severe inhibition of insulin signaling than either TSE-MVs or monocytes did alone. Most importantly, pretreatment of macrophages with CRM1 inhibitor III, before exposure to TSE, attenuated the ability of TSE-MVs released by these cells to impair insulin-induced phosphorylation of Akt (S473) in adipocytes.

Conclusion_
Our results indicate that exposure of human macrophages to smoke upregulates HMGB1 expression and causes its release on microvesicles, which mediate harmful crosstalk from macrophages to adipocytes, causing the MCP1 secretion by adipocytes, further recruitment of monocyte/macrophages, and impairments in insulin signaling that may contribute to insulin resistance and the development of T2DM in tobacco smoke exposure.
Abstract 170: Differential Effects of Atorvastatin and Digeranyl Bisphosphonate on Hemostasis and Thrombosis in Hypercholesterolemic Mice
Authors: Bhasin Neha , Stevens Jeff W , Niki Masaru , Tong Huaxiang , Kuder Craig H , Hohl Raymond J , Lentz Steven R 
Published: March 2022

Background and Hypothesis
Statins inhibit HMG-CoA reductase, leading to decreased production of cholesterol and other isoprenoids. There is growing evidence that statins have protective effects on arterial thrombosis through pleiotropic mechanisms that may be independent of their cholesterol lowering effects. The antithrombotic effects of statins have been proposed to be related in part to diminished synthesis of geranyl-geranyl pyrophosphate (GGPP). To test this hypothesis, we determined the hemostatic and thrombotic effects of atorvastatin and digeranyl bisphosphonate (DGBP), a specific inhibitor of geranyl-geranyl pyrophosphate synthase, in apolipoprotein E-deficient (ApoE-/-) mice.

Methods

Tissue levels of GGPP and its precursor farnesyl pyrophosphate (FPP) were measured by HPLC. ApoE-/- mice were treated with either vehicle, atorvastatin (50 mg/kg/d), or DGBP (0.4 mg/kg/d) subcutaneously for 7 days. Susceptibility to thrombotic occlusion of the carotid artery was measured in response to injury with FeCl
3
. Tail-transection bleeding time and platelet clot retraction were also assessed.

Results
Compared with vehicle- or atorvastatin-treated mice, DGBP- treated mice had elevated levels of FPP in heart (P<0.001) and lung (P<0.001) and decreased levels of GGPP in heart (P<0.01) and liver (P<0.01). The time to stable occlusion of the carotid artery was prolonged in atorvastatin-treated mice compared with vehicle-treated mice (8.6±2.4 vs. 5.3±0.3 minutes; P<0.05). In contrast, DGBP-treated mice were not protected from carotid artery thrombosis (6.8±0.9 minutes). Bleeding times were markedly prolonged in DGBP-treated mice (388±88 seconds) compared with atorvastatin- (98±11 seconds) or vehicle-treated (150±91 seconds) mice (P<0.05). Platelet clot retraction also was delayed in DGBP-treated mice compared with atorvastatin or vehicle-treated mice (P<0.05).

Conclusions
Inhibition of GGPP production causes impairment of hemostasis but does not prevent arterial thrombosis in hypercholesterolemic mice. The differential effects of DGBP and atorvastatin suggest that the antithrombotic effects of statins are independent of geranyl-geranyl-mediated processes.
Abstract 177: Pharmacological Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Intima Hyperplasia in Vein Grafts
Authors: Ji Yan , Weng Zhen , Strawn Tammy L , Fay William P 
Published: March 2022

Background.
Veins structurally and functionally remodel after being grafted into the arterial circulation. Many factors regulate vein graft (VG) remodeling, but the overall process is not adequately understood. We reported previously that genetic deficiency of plasminogen activator inhibitor-1 (PAI-1) enhances intimal hyperplasia (IH) of murine VGs. However, the effects of pharmacological inhibition of PAI-1 on VG remodeling are unknown.

Methods and Results.

We studied the effects of PAI-039, a small molecule that specifically inhibits free PAI-1, but not vitronectin (VN)-bound PAI-1, on VG remodeling. A segment of vena cava from a donor C57BL/6J mouse was inserted into the right carotid artery of a recipient mouse. Two groups were studied: 1) PAI-039 group, n=8; VG was coated with pluronic gel (50 μL) containing PAI-039 (25 μg/mL) during surgery; after surgery mice received PAI-039 (44 ug/kg by intraperitoneal [IP] injection) every other day for 3 weeks. 2) control group, n=8; mice received identical treatment, except that PAI-039 was omitted from pluronic gel and IP injection buffer. Four weeks later, mean intimal thickness and area were significantly less in PAI-039 treated mice than in controls (41.5±7.7 vs. 84.2±21.4 μm, and 0.17±0.044 vs. 0.34± 0.10 mm
2
, respectively,
P
<0.05). We also studied the effects of PAI-039 on proliferation of cultured venous smooth muscle cells (VSMC). PAI-039 (25 μM) significantly inhibited VSMC proliferation compared to vehicle control (0.65±0.073 vs. 0.82±0.078 AU [MTT assay],
P
<0.01).

Conclusion.
PAI-039, a specific pharmacological PAI-1inhibitor, inhibits neointima formation in VGs. This finding contrasts with our previously reported data indicating that genetic PAI-1 deficiency promotes VG IH. This paradox is most likely explained by the pleiotropic actions of PAI-1 and the capacity of PAI-039 to inhibit free PAI-1, which is motogenic, but not VN-bound PAI-1, which inhibits SMC migration. Regardless of mechanism, our results suggest that pharmacological PAI-1 inhibition may be a useful strategy to inhibit adverse VG remodeling and emphasize that mouse knock-out models do not necessarily predict the biological impact of pharmacological inhibitors.
Abstract 198: Myd88 Signaling Promotes Il6 Release and Slows Muscle Regeneration After Limb Ischemia
Authors: Sachdev Ulka , Cui Xiangdong , Tzeng Edith 
Published: March 2022

Introduction
MyD88 is an adapter protein involved in sensing tissue damage by innate immune receptors. We have previously shown that the absence of MyD88 protected against ischemia induced muscle injury in mice. This dramatic finding could be due to resistance to ischemia and/or accelerated regeneration. We hypothesize that MyD88 promotes ischemic muscle injury through increased inflammation and cell necrosis.

Methods
MyD88KO (KO) and WT littermates underwent femoral artery ligation (FAL) on the right hindlimb. Serum TNFα and IL6 were measured by ELISA at 24 hrs. After 1 wk, hindlimb muscle was examined for inflammatory cell infiltration, muscle necrosis, fat replacement, regenerating myocytes, and myofiber area (1 wk).

Results

After 24 h, serum TNFα levels were increased in mice undergoing FAL but were similar in KO and WT mice. Serum IL6, a myokine that inhibits muscle growth, was higher in WT than KO mice (79.3 ± 18.4 vs. 22.9 ± 11.8 pg/ml; p<0.05). One wk after FAL, inflammatory infiltration, fat replacement and muscle necrosis were extensive in both WT and KO mice. Regenerating myofibers, represented by multinucleated cells, were more numerous and larger in size in KO mice (148.7 ± 14.4 μm
2
vs. 77.3 ± 23.5 μm
2
, N=4-6 each; p=0.03; Fig 1).

Discussion
Ischemia promotes muscle inflammation and necrosis independent of MyD88. Instead, the absence of MyD88 enhanced regeneration, potentially through a reduction in IL6 production. Thus, contrary to our hypothesis, MyD88 appears to increase muscle injury following ischemia by inhibiting muscle regeneration and not through increased inflammation and necrosis.
Abstract 207: Comparison of Two Lines of Mice Expressing Smooth Muscle-Targeted Cre Recombinase from Different Promoters
Authors: Jaffe Mia , Du Liang , Dichek David 
Published: March 2022

Background
The development of mouse lines that drive inducible deletion of conditional alleles specifically in smooth muscle cells (SMC-Cre mice) is a major advance in experimental vascular biology. Two recently reported SMC-Cre lines differ in the promoter that drives Cre expression and in their sex linkage. One line uses a smooth muscle myosin heavy chain (SMMHC) promoter and is Y-chromosome linked; the other uses a smooth muscle actin (SMA) promoter and is autosomal. Performance of these lines has not been compared in the same laboratory.

Hypothesis
We hypothesized that the 2 lines of SMC-Cre mice would rearrange a floxed allele with equal efficiency and tissue specificity and would have equivalent_and at most low_levels of recombinase activity in vascular (V)SMC in the absence of Cre activation by Tamoxifen.

Methods
Both SMC-Cre lines were crossed with the R26R reporter line. Cre-positive/R26R-positive mice were injected with Tamoxifen or vehicle (n = 2 - 5/group). Cre-negative/R26R positive mice were injected with Tamoxifen (n = 3 - 4/group). Cre activity was assessed by X-gal stain (n = 2 - 4/group) and qRT-PCR for lacZ (n = 3 - 5/group).

Results
X-gal stains showed that both lines inducibly rearranged R26R with high efficiency in VSMC of the aorta and carotids. Visceral SMC in the bladder and gastrointestinal tissues of these mice also stained blue, as did VSMC within these organs and within other, SMC-poor organs such as skeletal muscle and heart. Vehicle-injected Cre-positive/R26R-positive mice in both lines had low levels of blue staining in VSMC but high levels of blue staining in SMC of the intestine (25 - 50%) and bladder (> 50%). No blue cells were detected in Cre-negative/R26R-positive mice injected with Tamoxifen. Both lines of Cre-positive/R26R-positive mice showed Tamoxifen-inducible expression of lacZ mRNA in the aorta, carotid, and bladder. Both lines also had essentially undetectable lacZ mRNA in the heart, either in the absence or the presence of Tamoxifen.

Conclusions
The 2 lines of SMC-Cre mice appear equivalent in their ability to achieve inducible gene deletion specifically in VSMC. Both have low levels of background activity in VSMC in the absence of Tamoxifen. Background Cre activity is higher in visceral than vascular SMC of both lines.
Abstract 213: Notch Activation in Aortic Adventitial Fibroblasts upregulates TGF-ß and may have Implications in Aortic Aneurysms
Authors: Liu Zhao-Jun , Li Yan , Velazquez Omaida C 
Published: March 2022

Objectives
Aortic adventitial fibroblasts (AoAF) are a rich source of cytokines and extracellular matrix (ECM). They critically participate in arterial wall remodeling and are implicated in the pathophysiology of aortic aneurysms (AA) and atherosclerosis. Little is known about signaling pathway(s) that regulate AoAF in cardiovascular diseases. Here, we examined and compared the status of Notch signaling in AoAF in human samples of normal, AA and atherosclerosis samples. We also explored the role of Notch signaling on AoAF cytokines and ECM production.

Methods
Immunohistochemistry was conducted to examine the Notch activity in AoAF of normal human aorta, segments with atherosclerotic plaque and segments of AA wall (N=4/type of aorta). Normal human AoAF were tested in vitro to study the effects of enforced Notch activation using lentiviral vector encoding the Notch1 intracellular domain, NIC/Lenti. The effect of Notch activation on cell proliferation was tested by MTT assay, and differentiation into myofibroblasts was tested by smooth muscle actin (α-SMA). The effect of Notch signaling on AoAF release of cytokines and ECM was studied using Pathway-Focused Human Fibrosis PCR Array (QIAGEN). Array findings were validated by Western blot.

Results
Compared to normal human aorta, significantly increased Notch activity was observed in AoAF from AA samples whereas decreased Notch activity was found in AoAF of atherosclerotic aorta (p<0.01). Notch activation inhibited cell proliferation (~30%, p<0.01) while induced differentiation of AoAF to myofibroblasts in vitro. Notch activation also resulted in a fibrotic response in AoAF as determined by increased expression of genes of collagen I/III, connective tissue growth factor (CTGF) and TGF-β1/2/3. Elevated expression of TGF-β 1/2/3(2.4-5.2-fold, p<0.001) in NIC/Lenti-transduced AoAF was confirmed by Western blot.

Conclusions
Notch signaling is activated in aortic adventitial fibroblasts at the site of aortic aneurysms and results in increased production of TGF-β by these cells.Our novel findings implicate a potential involvement of Notch-TGF-β signaling in the pathophysiology of aortic aneurysms.
Abstract 216: Profiling of Atherosclerotic Lesions by Tissue Microarrays Reveals Novel Proteins Mediating Disease Progression and Plaque Vulnerability
Authors: Perisic Ljubica , Hedin Erika , Razuvaev Anton , Mariette Lengqvist , Österholm Cecilia , Gillgren Peter , Ponten Fredrik , Odeberg Jacob , Hedin Ulf 
Published: March 2022

Objective
High-throughput immunohistochemistry (IHC) techniques are not commonly applied in studies of vascular diseases. Tissue microarrays (TMAs) are collections of multiple tissue cores placed in parallel in a single acceptor paraffin block, thereby enabling large-scale expression analyses of many samples concomitantly. Our aim was to evaluate this approach in qualitative and quantitative studies of candidate proteins implicated in atherosclerotic carotid stenosis.

Methods and Results
We constructed TMAs from a collection of 34 carotid plaques (6 asymptomatic/28 symptomatic patients) and assayed them for expression of 21 proteins by IHC. Majority of proteins chosen for the study were previously identified through microarray profiling of symptomatic vs. asymptomatic plaques (n=150) from the Biobank of Karolinska Endarterectomies. Quantification of stainings highlighted differential expression of several proteins in unstable compared to stable lesions (ADIPOR1, CD36, CD137, DOCK7), the most significantly upregulated being a secreted member of the proprotein convertase family, PCSK6 (p<0.0001). We localized this protein to smooth muscle actin positive cells and extracellular matrix of the fibrous cap in situ, overlapping with fibronectin and collagen IV. Endogenous PCSK6 localizes to cytosol and peripheral membrane extensions of human carotid smooth muscle cells (hcSMCs) in vitro. Increased expression of PCSK6 positively correlates to macrophages and lymphocytes markers in gene arrays, matrix degrading proteins (MMP9, ADAM9, Heparanase) and growth factors (PDGFB, TGF). Interestingly, stimulation of hcSMCs with several cytokines caused rapid upregulation of PCSK6 mRNA levels.

Conclusions
Here we demonstrate the feasibility of a scaled-up method for simultaneous and standardized expression studies in a large collection of human atherosclerotic lesions. Our results show that PCSK6, a novel protease previously not associated with atherosclerosis, is detected at increased levels in symptomatic carotid plaques, possibly in association with inflammation and extracellular matrix remodeling leading to plaque rupture. Further mechanistic studies are needed to dissect the role of PCSK6 in progression of atherosclerosis.
Abstract 224: The Tyrosine Kinase Arg Mediates Endothelial Barrier Disruption by Modulating Integrin Distribution
Authors: Van Nieuw Amerongen Geerten P , Aman Jurjan , van Bezu Jan , Valent Erik , van Hinsbergh Victor 
Published: March 2022

Introduction
Endothelial barrier disruption and vascular leakage during inflammation forms an important threat to the critically ill. In a recent study we demonstrated that the tyrosine kinase inhibitor imatinib effectively attenuates endothelial barrier disruption via inhibition of the kinase Abl-related gene (ARG). Here, we evaluate how ARG mediates endothelial barrier disruption.

Methods & Results

ARG was depleted from endothelial cells with various siRNAs, yielding >85% reduction in ARG protein expression. Disruption of the endothelial barrier by the inflammatory mediator thrombin was lower in ARG-depleted cells than in control cells (30% versus 60% drop in endothelial resistance). ARG deficient mice showed a significant reduction of VEGF-induced vascular leakage, stressing the
in vivo
contribution of ARG to vascular leakage. ARG was activated upon endothelial activation
in vitro
, as indicated by a 2-3 fold increase in CrkL phosphorylation, the downstream target of ARG.

Surprisingly, ARG depletion resulted in 40% reduction in VE-cadherin protein expression. At the same time, ARG-depleted cells completely lacked a contractile response to inflammatory mediators, despite normal myosin light chain phosphorylation. ARG depletion resulted in increased protein expression of α
5
β
1
integrin, increased expression of β
1
integrin at the cell surface and redistribution of α
5
β
1
integrin to the cell edges (close to cell-cell contacts). These signaling events were associated with increased adhesive force of ARG-depleted endothelial cells to the extracellular matrix. The clinical relevance of ARG was confirmed by increased amounts of phosphorylated CrkL in the pulmonary endothelium of septic patients.

Conclusion
This study shows that ARG is a significant mediator of endothelial barrier disruption during inflammation. ARG regulates integrin dynamics during endothelial barrier disruption, and the ARG-induced changes in integrin dynamics contribute to endothelial barrier disruption.