bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Abstract 239: Dpp-4 Inhibitor Alogliptin Prevents Abdominal Aortic Aneurysm Formation and Development in Rat Model
Authors: Morimoto Keisuke , Bao Wulan , Yu Zhenhai , Jie Yu , Sasaki Naoto , Yamashita Tomoya , Hirata Kenichi , Okita Yutaka , Okada Kenji
Published: March 2022

Objective

Dipeptidyl peptidase-4 (DPP-4) inhibitor, a novel anti-diabetic drug, has been proved to have a cardioprotective effect on ischemia-reperfusion injury through an antioxidant effect. Although we reported reactive oxygen spices (ROS) play a crucial role in AAA formation and development, the effect of DPP-4 inhibitor on AAA has not been investigated. In this study, we examined the effect of DPP-4 inhibitor alogliptin on experimental AAA rat model.

Methods

An AAA model induced with intraluminal elastase and extraluminal calcium chloride was created using 48 rats. Thirty-six rats were divided into 3 groups; a low dose (group LD; 1mg/kg/day), high-dose (group HD;3mg/kg/day), and control (group C; pure water). Alogliptin was administered by gastric gavage once per day beginning 3 days before surgery. The remaining 12 rats received a delayed alogliptin administration (group DA-LD; 1mg/kg/day, group DA-HD; 3mg/kg), starting on day 7 after aneurysm preparation up to 28 days. On day 7, ROS expression was semi-quantified by dihydroethidium staining and the oxidative product of DNA induced by ROS, 8-hydroxydeoxyguanosine (8-OHdG), by immunohistochemical staining. Histopathological examination was performed on day 28, and AAA dilatation ratio was calculated.

Results

On day 7, ROS expression were reduced (4.6±0.6 in group C, 2.7±0.3 in group LD, 1.7±0.5 in group HD; p<0.001) and there were fewer 8-OHdG positive cells in alogliptin treated samples (138.1±7.4 in group C, 102.5±4.5 in group LD, 66.1±4.5 in group HD; p<0.001). The treatment reduced messenger RNA expression and activity of matrix metallo-proteinases 2 and 9 in aneurysm walls. On day 28, aortic walls in group LD and HD were less dilated than those in group C (199.2±11.8 % in group C, 159.6±2.8 % in group LD, 147.1±1.9 % in group HD; p<0.02). Delayed alogliptin administration prevented further aneurysm dilatation compared with the aortic walls in group C at day 7 (158.9±8.5 % in group C at day 7, 160.1±14.3 % in group DA-LD, 155.1±2.6 % in group DA-HD; n.s.).

Conclusion

DPP-4 inhibitor alogliptin treatment starting on day 7 after aneurysm preparation completely inhibited aneurysm development. Alogliptin could be effective pharmaceutical agent against AAA in clinical practice.
Abstract 242: Novel Therapeutic Approach to Abdominal Aortic Aneurysm through the Inhibition of Osteoclastogenesis
Authors: Takei Yuichiro , Kurosawa Koji , Yamanouchi Dai
Published: March 2022

Arterial calcification, which mainly consists of calcium phosphate (CaPO4) crystals, has been proven to be an organized process, just like the processes seen in bone. Osteoclast-like cells (OCLCs), which are derived from the monocyte/macrophage lineage, dissolve extracellular matrices with proteases. Although OCLCs have been reported to exist in the calcified artery, their role in abdominal aortic aneurysm (AAA) hasn’t previously been explored and was investigated in this work.

First, we obtained human aortic tissues from patients who had undergone surgical repair for AAA and aortic stenotic disease (N = 5). Calcifications were abundant in both groups. However, multinucleated cells positive for osteoclast markers (enzymatic tartrate-resistant acid phosphatase staining, calcitonin receptor, vitronectin receptor, and CD68) were identified only in AAA tissues. We then checked the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), key regulators of osteoclastogenesis, and TNFα, a pro-inflammatory cytokine. Immunohistochemical analysis showed upregulation of TNFα but not RANKL/OPG in AAA compared with stenosis. To mimic the pathogenic conditions of aneurysm and stenotic disease in vitro, we stimulated macrophages (RAW 264.7 cell line) with CaPO4 crystals with or without TNFα (100 ng/ml). Aneurysmal conditions (CaPO4 with TNFα) induced a significantly greater number of OCLCs than stenotic conditions (CaPO4 without TNFα) or control conditions (TNFα only, P < 0.01), with higher MMP-9 secretion after 5 days. Furthermore, inhibition of OCLCs with anti-TNFα (10 μg/ml, locally) or bisphosphonate (zoledronate, 100 μg/kg, i.v.) inhibited mouse CaPO4 aneurysm and osteoclastogenesis (P < 0.01, N = 5). Finally, we confirmed a spatial correlation between fluorescein-conjugated bisphosphonate and OCLCs in the CaPO4-treated artery.

In conclusion, we have demonstrated the existence of OCLCs in AAA and therapeutic effects of OCLCs inhibition on aneurysms. Our in vitro data showed that TNFα with CaPO4 induces osteoclastogenesis. These data suggest that inflammation in the calcified artery initiates osteoclastogenesis, which may be used to differentiate between stenotic and aneurysmal degeneration.
Abstract 244: Targeted Deletion of Transcription Factor BATF Reduces Experimental Abdominal Aortic Aneurysm Formation and Progression
Authors: Xu Baohui , Xuan Haojun , Tanaka Hiroki , Wang Wei , Fujimura Naoki , Glover Keith J , Xie Weiying , Xiong Xiaoxing , Zhao Heng , Michie Sara A , Dalman Ronald L
Published: March 2022

Transcription factor BATF, a regulator of T cell IL-17-production and B cell immunoglobulin class switching, may be relevant in abdominal aortic aneurysm (AAA) disease pathogenesis. This study compared AAA formation following porcine pancreatic elastase (PPE) infusion in C57BL/6 and BATF-deficient mice. In wild-type mice, message for BATF and IL-17A was highly expressed in aneurysmal compared to control (PBS infusion) aortae. In BATF-deficient mice, aortic diameter enlargement following PPE infusion was almost completely suppressed, with preservation of medial elastin and smooth muscle cellularity, and attenuation of transmural aortic angiogenesis, leukocyte accumulation, and IL-17A-expressing CD4 T cells in peri-aortic lymph nodes and the spleen. AAA formation in response to PPE-infusion was rescued in BATF-deficient mice via either parenteral adminstration of IL-17A or wild-type murine leukocytes. In conclusion, BATF is essential for aneurysm formation following intraluminal aortic PPE infusion in C57BL/6 mice. BATF, IL-17A, or related T- and B-cell immunologic mediators may represent rational therapeutic targets for suppression of clinical AAA disease.
Abstract 245: Metabolic Syndrome and Common Carotid Artery Remodeling: Study of Women’s Health Across the Nation
Authors: Asubonteng Julius O , Barinas-Mitchell Emma J , Wisniewski Stephen R , Selzer Faith M
Published: March 2022

Objective

The metabolic syndrome (MetS) is linked to a higher risk of cardiovascular disease (CVD) events, including stroke. Measures of maladaptive carotid artery remodeling such as greater common carotid artery (CCA) intima-media thickness (IMT), and wider adventitial diameter (AD) are more prevalent among older women, varies by race/ethnicity and is predictive of stroke, however, data are lacking in women during the menopausal transition. Thus, we hypothesized more maladaptive carotid remodeling among MetS than those without MetS by race/ethnicity.

Methods

Study of Women’s Health Across the Nation (SWAN) participants, free of clinical CVD and with IMT and AD data at the 12th annual visit were evaluated. Lumen diameter (LD), circumferential wall stress (CWS) and circumferential wall tension (CWT) were calculated. Multivariable linear regressions were used to investigate the relationship between MetS and CCA parameters in all women and by race/ethnicity. Covariates included were age, height, site, LDL-c, education, medication use, menopause status and smoking.

Results

A total of 1183 women (41.3% MetS; 96.9% Postmenopausal; 52.1% Caucasian, 27.6% African American, 14.6% Chinese, 5.8% Hispanic) from 6 sites across the USA, aged 60.1±2.7 years old at visit 12 were included. MetS was associated with a larger LD (β(SE)): 0.222(0.033) mm, P <0.0001; AD (0.317(0.038)) mm, P <.0001; IMT(0.047(0.006)) mm, P <.0001; CWS (2.51(0.938)) kPa, P <.0077; CWT (5.53(0.637) kPa, P <.0001), after adjusting for covariates. Compared to Caucasians, LD was greater in both African American and Chinese ( (0.130 (0.040) mm, P <.0001; 0.292(0.064) mm, P<.0001, respectively), AD was greater in African American ((0.236(0.046) mm, P <.0001) and Chinese ((0.331(0.073) mm, P <.0001), a thicker IMT((0.053(0.008) mm, P <.0001), CWT was greater for African American and Chinese ((4.83(0.776) kPa, P <.0001; 3.18(1.22) kPa, P =0.0093).

Conclusion

MetS is associated with maladaptive remodeling of the CCA, as evidenced by larger AD, LD, IMT and CWT. Importantly, the association between MetS and CCA remodeling varies between race/ethnicity, which could partially explain differences in stroke rates by race.
Abstract 25: Platelet Activation by GPVI, GPIb-V-IX and aIIbß3 Requires PLC?2 but Not PLC?1
Authors: Zheng Yongwei , Zhi Huiying , Adams Tamara , Wen Renren , Newman Peter J , Wang Demin , Newman Debra K
Published: March 2022

Receptor-mediated cellular activation requires phospholipase C (PLC) activity to generate second messengers that elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are grouped into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2), which are critical for cellular activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2; however, their relative roles in platelet activation are poorly understood. Receptors that use PLCγ activity to activate platelets include the glycoprotein (GP) VI collagen receptor, GPIb-V-IX receptor for von Willebrand Factor (VWF), and integrin αIIbβ3 receptor for fibrinogen (Fg). While PLCγ2 is critical for platelet responses to GPVI-specific stimuli and in VWF-dependent thrombus formation, its role in αIIbβ3-dependent responses is controversial, suggesting that PLCγ1 may compensate for PLCγ2, when absent, in αIIbβ3-mediated outside-in signaling. Assessment of the role of PLCγ1 in platelet activation is hindered by the early embryonic lethality of PLCγ1-deficient (PLCγ1-/-) mice. We obtained platelets from newly-generated conditionally PLCγ1-/- mice and compared their responses to αIIbβ3-, GPVI-, and GPIb-V-IX-specific stimuli with those of platelets from wild-type (WT), PLCγ2-/-, and PLCγ1-/-/PLCγ2-/- mice. We found that platelet spreading on immobilized Fg, collagen-induced aggregation, and VWF-dependent thrombus formation are severely impaired in PLCγ2-/- or PLCγ1-/-/PLCγ2-/- mice. Defective spreading on immobilized Fg was overcome by addition of exogenous ADP, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistent with this hypothesis, αIIbβ3-mediated granule release was impaired in the absence of PLCγ2. In contrast, platelets from PLCγ1-/- mice spread and released granule contents normally on Fg, exhibited near normal levels of GPVI-dependent platelet aggregation, and formed near normal thrombi over immobilized collagen/VWF. These results establish that PLCγ2 is indispensable for platelet secretion downstream of adhesive interactions mediated by GPVI, GPIb-V-IX, and αIIbβ3, and demonstrate that PLCγ1 plays little role in these processes.
Abstract 26: 12-Lipoxygenase is an Essential Regulator of Fatty Acid Protection Against Platelet Activation and Thrombosis
Authors: Yeung Jennifer , Vesci Joanne , Holman Theodore R , Holinstat Michael
Published: March 2022

Introduction

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of platelet function by reacting with arachidonic acid (AA). It is possible that a number of other fatty acids present on the platelet surface are oxidized by 12-LOX. Identifying novel bioactive metabolites which regulate platelet function are essential for understanding the role of 12-LOX in regulation of thrombosis, vessel occlusion, and stroke.

Objectives

We sought to characterize the substrate specificity of 12-LOX against several polyunsaturated fatty acids (PUFAs) in vitro, ex vivo, and in vivo and determine if PUFA metabolites act as endogenous regulators of platelet activation.

Methods

Metabolite production was measured in vitro by assessing which PUFAs are oxidized by 12-LOX to form bioactive metabolites. These metabolites were further characterized for anti-platelet properties in platelets ex vivo. Finally, using wild-type and 12-LOX-deficient mouse models, the importance of 12-LOX in regulating the protective effects of these PUFAs was assessed in vivo.

Results

A number of PUFAs were found to be oxidized by 12-LOX in vitro. The bioactive lipid products resulting from 12-LOX oxidation of DGLA in particular, 12-(S)-hydroperoxy-8- Z, 10E, 14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, α IIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited platelet activation as well as platelet clot retraction. Blocking 12-LOX rescued normal platelet activity. Finally, mice fed a DGLA-enriched diet showed increased bleeding and decreased platelet activity. However, mice deficient in 12-LOX were resistant to DGLA-induced attenuation of platelet activation but not direct application of 12-HETrE.

Conclusions

These observations support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane. DGLA and its metabolite 12-HETrE may help explain the protective effect of PUFAs on cardiovascular function.
Abstract 265: Perioperative Silent Myocardial Ischemia, the Use of Blood Transfusions and Their Associations With Long-Term Mortality Following Peripheral Vascular Surgery
Authors: Schneider Gabriel , Rockman Caron , Riegel Daniel , Berger Jeffrey
Published: March 2022

Objective

Thrombotic and bleeding complications during the perioperative period are a major concern during the postoperative period. However, the relationship between perioperative myocardial necrosis and major bleeding with long-term mortality is uncertain.

Methods

In a retrospective study, we analyzed data from 289 consecutive patients during a one-year period between 2009-2010 who underwent open major vascular surgery (carotid endarterectomy, above/below knee amputation, open aneurysm repair, extremity bypass, and embolectomy). Myocardial necrosis was defined as any cardiac troponin T value ≥ 0.1 ng/ml and major bleeding was defined as requiring ≥2 units of blood within 24 hours of the operation. Lower threshold cutoffs (troponin >0.04 ng/ml and any transfusion) were also investigated. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression.

Results

During a mean follow-up period of 1.8 years, there were 46 deaths (15.9%). Myocardial necrosis and major bleeding occurred in 25.2% and 14.9% of subjects, respectively. After adjustment for age, sex, smoking, hypertension, diabetes and coronary artery disease, both myocardial necrosis (OR 4.24, 95% CI 1.82-9.87) and major bleeding (OR 2.56, 95% CI 1.28-5.46) were associated with long-term death. Similarly, lower threshold cutoffs of troponin >0.04 ng/ml and any transfusion independently predicted a 6.27-fold and a 6.44-fold increase in long-term mortality (p<0.001 for both).

Conclusions

Perioperative myocardial necrosis and major bleeding, even at low cutoff levels, are independent and complementary predictors of long-term mortality following major vascular surgery.
Abstract 272: Epidermal Growth Factor Dependent Collagen Loss in Human Carotid Plaques is Mediated by Matrix Metalloproteinase-9
Authors: Rao Velidi H , Kansal Vikash , Hunter William J , Agrawal Devendra K
Published: March 2022

Aims

Atherogenesis is a chronic progressive process that develops over several decades. Changes in the structure and composition of extracellular matrix (ECM) play an important role in the process of atherosclerosis. Matrix metalloproteinases (MMPs) and growth factors present in atherosclerotic human plaques could be related to plaque remodeling and increased risk for plaque destabilization and rupture.

Hypothesis

Selective inhibition of elevated MMP-9 and epidermal growth factor receptor (EGFR) increases fibrillar collagens (Type I and III) in vascular smooth muscle cells (VSMCs) from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis.

Methods and results

Cultured human VSMCs from both AS and S patients were treated with or without epidermal growth factor (EGF). The proteolytic activity of MMP-9 and MMP-2 was quantified by gelatin zymography. The mRNA transcripts of MMP-9, MMP-1, EGFR and collagen types I (COL1A1 and COL1A2) and III (COL3A1) were analyzed by qPCR. EGF treatment significantly increased MMP-9 activity and mRNA transcripts for MMP-9, MMP-1and EGFR, while mRNA transcripts for collagen types I and III were decreased in both AS and S groups. Collagen type I and type III mRNA transcripts were significantly decreased in symptomatic VSMCs compared to cells from AS. Inhibition of EGFR with AG1478 and MMP-9 using small molecule inhibitor and transfection with MMP-9 siRNA decreased the activity and mRNA expression of MMP-9 and increased mRNA transcripts of collagen I and III in EGF treated VSMCs.

Conclusion

These results reveal a novel mechanism by which MMP-9 induced by EGFR activation could be a leading cause of plaque instability in patients with carotid stenosis; and the selective blockade of EGFR and MMP-9 may be a novel strategy and a promising target for treating human unstable and vulnerable plaques.
Abstract 275: Cholesterol Efflux Capacity is Inversely Associated with Severity of Carotid Atherosclerosis and Increases with Time Since Cerebrovascular Event
Authors: Doonan Robert-James , Hafiane Anouar , Genest Jacques , Daskalopoulou Stella S
Published: March 2022

Introduction

HDL is thought to exert its atheroprotective role by promoting cholesterol efflux from lipid-laden macrophages. Cholesterol efflux capacity (CEC) has been shown to be inversely associated with carotid intima-media thickness and presence of coronary artery disease. We assessed the hypothesis that CEC is associated with severity of carotid atherosclerosis and with cerebrovascular symptomatology.

Methods

Symptomatic (n=114) and asymptomatic (n=41) patients with carotid stenosis were recruited from Vascular Surgery at the Royal Victoria and Jewish General hospitals, Montreal, Canada. Carotid Doppler ultrasound was performed and stenosis (50-79%, 80-99%) was graded according to velocities. Detailed information on symptomatology obtained. A blood sample was collected on the day of the ultrasound; HDL was obtained by polyethylene glycol precipitation after depletion of apoB-containing lipoproteins. CEC was determined by incubating HDL in cAMP-stimulated J774 mouse peritoneal macrophages for 6 hours. Specific cholesterol efflux was obtained by subtracting total efflux from efflux in non-cAMP stimulated cells. Differences in CEC were assessed using linear regression according to 1) stenosis, 2) symotomatology and, 3) timing of symptomatology.

Results

Compared to patients with 50-79% stenosis (n=31), patients with 80-99% stenosis (n=124) had significantly decreased CEC (beta=-2.23, P=0.04) after adjustment for age, sex, apoAI, and systolic BP. CEC was not significantly different between symptomatic or asymptomatic patients. However, in symptomatic patients CEC increased with increasing time since cerebrovascular event. Specifically, compared to 0-30 days (n=72), CEC was non-significantly increased 31-90 days since event (n=31, beta=1.64, P=NS), while increased significantly ≥ 90 days since event (n=11, beta=4.48, P=0.01), after adjustment as described above.

Conclusion

These results suggest that CEC is inversely associated with severity of carotid stenosis and that CEC increases with increasing time since symptomatic event. This may be related to remodeling of HDL during the acute phase reaction after a recent ischemic event.
Abstract 292: Magnetic-based Multi-layer Microparticles for Stem Cell Isolation, Enrichment, and Detachment
Authors: Wadajkar Aniket S , Santimano Sonia , Menon Jyothi , Nguyen Kytai T
Published: March 2022

Due to the extensive therapeutic potential of endothelial progenitor cells (EPCs) in regenerative medicine, various techniques have been developed to isolate and expand them
ex vivo
. Yet these methods are limited by the use of harmful chemicals and difficulty in
ex vivo
stem cell expansion. The aim of this research was to develop multi-layer microparticles (MLMPs) that can magnetically isolate EPCs without using Ficoll and harsh shear forces, provide sequential proliferating and differentiating growth factor release for stem cell enrichment, and detach cells in response to temperature changes without the use of chemicals such as Trypsin. MLMPs consisted of a poly(lactic-co-glycolic acid) (PLGA) core embedded with magnetic nanoparticles and the temperature-sensitive polymer (poly(
N
-isopropyl acrylamide-allylamine): PNIPAAm-AH) shell. MLMPs were formulated and characterized for their physicochemical properties. Studies to investigate growth factor release, optimization concentrations of MLMPs and antibodies as well as the time required for maximal cell isolation were also carried out. Finally, effects of MLMPs on the growth of isolated cells were studied. MLMPs had core-shell structure with spherical morphology of 50-75 μm, and their growth factors were released in a sequential manner required for cell proliferation and differentiation. EPCs successfully attached to the polymer surface, while maintaining their morphology as verified from staining and SEM. Moreover, 64% EPCs were isolated over a period of 2 hours with 1 mg/ml MLMPs conjugated with 3 μg/ml CD34 antibodies. Cells were successfully detached from the particle surface by lowering the surrounding temperature to room temperature for a short time. MLMPs loaded with growth factors show an enhanced cell growth compared to those of commercial beads. These results indicate that MLMPs might have a significant impact on cell isolation and expansion technologies.
Abstract 299: Cell Division Cycle 7 Mediates Transforming Growth Factor-ß-induced Smooth Muscle Maturation
Authors: Shi Ning , Chen Shi-You
Published: March 2022

Cell division cycle 7 (Cdc7) has been shown to regulate cell fate determination in the initial phase of transforming growth factor-β (TGF-β)-induced smooth muscle cell (SMC) differentiation. It is unknown, however, if Cdc7 is involved in SMC maturation or the late phase differentiation. In this study, we found that Cdc7 regulates the late phase SMC differentiation through a mechanism different from the cell fate determination. Knockdown of Cdc7 suppresses TGF-β-induced expression of smooth muscle myosin heavy chain (SMMHC), a late marker of SMC differentiation in addition to the early markers. Cdc7 overexpression, on the other hand, enhances SMMHC expression. Interestingly, Cdc7 activates the mRNA expression and gene transcription of myocardin (Myocd), a master regulator for SMC differentiation, whose transcription is blocked in the initial phase of the differentiation because TGF-β does not induce Myocd mRNA until 16 h after the stimulation. These data suggest that Cdc7 mediates TGF-β-induced late SMC differentiation via activating Myocd. Mechanistically, Cdc7 physically and functionally interacts with Nkx2.5 to regulate Myocd promoter activity. Cdc7 appears to enhance Nkx2.5 binding to Myocd promoter, leading to Myocd activation. Taken together, our studies demonstrate that Cdc7 regulates the initial and late phase of SMC differentiation through distinct mechanisms.
Abstract 306: Transfected Early Growth Response Gene-1 DNA Enzyme Attenuates Autogenous Vein Graft Intimal Hyperplasia in vivo
Authors: Liu Chengwei , Wang Shi , Zhang Xuesong , Peng Yu , Tao Ming , Ozaki Charles Keith , Wang Shuqing
Published: March 2022

Introduction

Early growth response gene-1 (Erg-1) functions centrally in cell proliferation. We hypothesized that introduction of a novel Egr-1 DNA enzyme (EDRz) via liposomes into the vein graft would attenuate occlusive SMC proliferation and intimal hyperplasia.

Methods

Rat jugular vein was anastomosed to the infrarenal abdominal aorta end-to-end. EDRz (n=90) was applied to the graft vein adventitia (no therapy control; n=90). Animals were harvested at 1, 2, and 6hr, and 1, 3, 7, 14, 28, and 42d (10 EDRz/10 control per time point). Analyses included light microscopy (morphology), fluorescence (EDRz transfection), RT-PCR and
in situ
hybridization (Egr-1 mRNA), and Western blot and immunohistochemistry (Egr-1 protein).

Results

EDRz localized to the vein graft media early; Egr-1 protein was in medial SMCs, monocytes, and ECs; treated grafts had no detectable Egr-1 RNA or protein by 7d. VSMC proliferation (p<0.01 from 3-42d) and intimal thickness were attenuated by EDRz (p<0.01 from 7-42d; Fig ).

Conclusions

EDRz reduces Egr-1 expression, SMC proliferation, and intimal hyperplasia in autogenous vein grafts. EDRz offers a novel approach to enhance vein graft durability.

Figure. The result of thickness of intimal hyperplasia in vein graft and EDRz transfection after

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Abstract 313: Phosphorylation of Runx2 and Osteochondrogenic Differentiation of Vascular Smooth Muscle Cells
Authors: Chen Theodore M , Nguyen Ngoc B , Eaton Karen V , Ge Chunxi , Franceschi Renny T , Giachelli Cecilia M , Speer Mei Y
Published: March 2022

Vascular calcification, the abnormal deposition of calcium phosphate salts in the vasculature, is associated with a heightened risk of cardiovascular disease in patients with chronic kidney disease and diabetes mellitus. Through a genetic fate mapping strategy, our laboratory has identified vascular smooth muscle cells (SMCs) as a major cell source contributing to bone- and cartilage-like cells in calcifying arteries. Runx2, a transcription factor critical for osteoblast and hypertrophic chondrocyte differentiation, is upregulated in the early stages of a wide range of calcific arterial diseases. Treating SMCs
in vitro
with elevated phosphate resulted in an increase in phosphorylated Runx2 at serine 319, while total Runx2 remained unaffected. We also found that ERK1/2 signaling and Runx2 were required for SMC osteogenic differentiation and calcification
in vitro
. To further explore whether Runx2 phosphorylation and activation are critical to Runx2 function in vascular calcification, we mutated Runx2 at serine 301 and 319, the sites phosphorylated by active ERK and shown to be crucial for Runx2 transcriptional activity in osteoblasts, to create ERK-resistant (S310A/S319A) and constitutively active (S301E/S319E) variants. We have generated Runx2 knockout SMCs from Runx2-
flox/flox
mice carrying SM22α-Cre recombination transgenic allele. Wild type Runx2 and its mutants were introduced into these cells retrovirally. Preliminary Runx2 functional analyses suggest a successful introduction of the constructs and a phosphate-dependent activation of Runx2. Experiments to address a role of Runx2 phosphorylation by ERK in SMC osteogenic differentiation and matrix calcification are ongoing. In conclusion, our current findings along with the ongoing experiments will provide important inside into mechanisms of SMC osteochondrogenic differentiation and vascular calcification, findings that may serve as basis for the development of appropriate therapeutic strategies for this devastating complication.
Abstract 316: A Superantigen Whole Blood Challenge Test for the Quantification of Immuno-inflammation in Early Phase Drug Development
Authors: van Poelgeest Eveline , Burggraaf Jacobus , Malone Karen , Kluft Cornelis , Moerland Matthijs
Published: March 2022

Development of immune-modulating drugs is challenging as assessment of intended pharmacology in healthy volunteers is difficult. Commonly, ex-vivo LPS challenges of whole blood or isolated cells are applied. However, the applicability of the LPS challenge is limited as LPS primarily stimulates germ-line encoded innate-immune TLR4-receptors, and thus does not allow monitoring of pharmacological modulation of the adaptive immune compartment. We explored an alternative whole blood challenge, inducing lymphocyte-driven inflammation by the superantigen staphylococcal enterotoxin B (SEB), and compared this to LPS. Further validation was performed by assessing the inhibitory effects of different immune-modulating compounds (MAP kinase inhibitors, calcitriol, methotrexate, protein kinase C, doxycycline, simvastatin), allowing discrimination between effects on monocytes and lymphocytes.

SEB induction resulted in the release of a cytokine panel that well-resembled cytokine release observed after physiological lymphocyte stimulation, with clear induction of IL-2, IFN-γ, TNF- α, IL-10 and GM-CSF. Based on TNF-α and IL-2 release, SEB’s maximal effect was reached at 100 ng/mL. Whereas inter-individual variability in EC50 was relatively small for LPS (0.32±0.10 ng/mL), for SEB inter-subject variability was distinctly higher (62±54 ng/mL). MAP kinase inhibitors and calcitriol were effective in inhibiting SEB-induced TNF-α and IL-2 release, demonstrating an inhibitory effect on the monocyte and/or lymphocyte. Importantly, whereas methotrexate, doxycycline, and simvastatin did not affect SEB-induced TNF-α release, a clear inhibiting effect on SEB-induced IL-2 release was observed, implying a direct action of these compounds on the lymphocyte.

These data demonstrate that the SEB challenge can be used to estimate the immune-modulating effects of compounds directly acting on the T-lymphocyte population. As such, the whole blood SEB challenge is a useful translational tool in early drug development, especially since immune-inflammation is a target in the development of new treatment modalities for currently poorly-controlled diseases such as rheumatoid arthritis and psoriasis, and cardiovascular disease.
Abstract 317: A Congenic Mouse Model for Carotid Intima-Media Thickening Demonstrates that Leukocyte Infiltration is a Genetically Regulated Trait
Authors: Smolock Elaine M , Korshunov Vyacheslav A , Berk Bradford C
Published: March 2022

We demonstrated that inflammation and intima-media thickening (IMT) are increased in carotids exposed to low blood flow in the SJL/J (SJL) mouse strain compared to other strains. We identified three novel quantitative trait loci (QTLs) on chromosomes (chr) 2, 11, and 18 that control IMT in a genetic cross between C3HeB/FeJ (C3H/F) and SJL mice. Using a genetic backcross of C3H/FxSJL we measured inflammation in carotid IMT as a quantitative trait. Immunostaining for CD45+ (a pan-specific leukocyte marker) was performed on carotids from C3H/F, SJL, F1 and N2 progeny to measure leukocyte infiltration. A QTL for CD45+ cell infiltration was identified on chr11 (17cM, LOD=2.3). Interval mapping showed that the CD45+ locus accounted for 8% of the variation in the C3H/FxSJL backcross. Importantly, the CD45+ locus co-localized with our previously reported intima modifier 2 (Im2) locus. We created two Im2 congenic mice (C3H/F.SJL.11.1 and C3H/F.SJL.11.2) to define the contribution of chr11 to IMT. The C3H/F.SJL.11.1 congenic mouse showed elevated CD45+ staining in the vascular wall in response to low flow. Thus, the CD45+ trait partially explains the intima trait. This reveals a potential mechanistic relationship between leukocyte infiltration and IMT in response to decreased blood flow.