bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dietary nitrate suppresses platelet reactivity in Apolipoprotein E Knockout Mice
Authors: Filomena Federica , Khambata Rayomand , Ahluwalia Amrita 
Published: November 2023

INTRODUCTIONReduced endothelium‐derived NO bioavailability is a characteristic of the endothelial dysfunction that occurs in the early stages of atherogenesis and is thought to account in part for the enhanced platelet activity that characterises this disease state. Dietary supplementation with inorganic nitrate (NO3−) has been proven to suppress ex vivo platelet activation in healthy volunteers [1, 2] and to reduce platelet activation in hypercholesterolemic patients [3]. This effect relies on the enterosalivary circulation, where the oral microbiome converts NO3− to NO2−, resulting in increased circulating NO2− levels. This NO2− is then converted by mammalian vascular nitrite reductases to NO, which, amongst the other beneficial effects on the vasculature, appears to reduce platelet activation. However, whether dietary NO3− might be an effective antithrombotic agent for therapeutics in atherosclerosis is unknown.AIMTo investigate whether dietary supplementation with inorganic NO3− reduces platelet activity and thus thrombotic potential in vivo in the apolipoprotein E (ApoE) knock out (KO) mouse model of atherosclerosis.METHODSMales or female, 4/6 week old, ApoE KO or wildtype (WT, C57BL6) littermate mice were supplemented with 15mM KNO3 or 15mM KCl in the drinking water for 10 days. Thrombotic potential was assessed in vivo using intravital microscopy following i.v. injection of the platelet marker DyLight488‐labelled CD42 antibody. A 10% FeCl3 solution was applied for 1 min on a mesenteric artery and thrombus formation recorded for 45 min. Tail bleeding time after excision of 0.25cm from the tail tip was also assessed with standard methods. Blood was collected and platelet counts determined using counting beads. Data are shown as mean±s.e.m.RESULTSKNO3 feeding raised plasma levels of NO3− and NO2− compared to KCl as assessed using the method of ozone chemiluminescence ([NO2−]μM ApoE KO: KCl=0.6±0.07 KNO3=0.8±0.06, WT: KCl=0.8±0.1 KNO3=1.0±0.1. [NO3−]μM ApoE KO: KCl=17.3±3.2 KNO3=68.1±17.8, WT: KCl=34±4.6 KNO3=100.4±24.1. n=6). Treatment with KNO3 suppressed FeCl3‐induced arterial thrombosis in both WT (n=3) and ApoE KO (n=7) animals (Figure 1). A trend towards a prolonged bleeding time was observed in all mice receiving KNO3, although this did not reach statistical significance (ApoE KO: KCl=3.9±1.2min KNO3=6.6±1.1min, WT: KCl=4.1±1min KNO3=4.9±1min). Platelet count was not different between treatment and control groups (ApoE KO: KCl=2.8×105±0.7×105 KNO3=3×105±0.7×105; ApoE WT: KCl=2.8×105±0.4×105 KNO3=2.8×105±0.7×105).CONCLUSIONSWe have shown that supplementation with dietary NO3− in the setting of atherosclerosis modestly reduces in vivo platelet reactivity and thus thrombotic potential. Dietary nitrate could represent a cheap and effective strategy to dampen platelet activity in patients at high risk of thrombosis.Support or Funding InformationFF is supported by a MRC PhD Studentship.
STIM1 deficiency in VSMC promotes vascular calcification in atherosclerosis
Authors: Yang Youfeng , Sun Yong , Chen Yabing 
Published: November 2023

ObjectiveVascular calcification is a characteristic feature of atherosclerosis. We and others have demonstrated that osteogenic differentiation of vascular smooth muscle cells (VSMC) contribute predominantly to the pathogenesis of vascular calcification in atherosclerosis. The key cellular event that leads to calcification is the secretion of matrix vesicles (MVs), membrane‐bound nanoparticles that carry calcium and matrix proteins to initiate calcification. However, the molecular regulation of MV release and the causal effect of MV release on VSMC calcification are poorly understood. The objective of this study is to investigate the function of a key calcium flux regulator, stromal interaction molecule 1 (STIM1), in regulating MV release and VSMC calcification; and to elucidate the underlying molecular mechanisms.MethodsSMC‐specific STIM1‐deficient mice (STIM1Δ/Δ SMC) were generated by breeding smooth muscle specific SM22α‐Cre mice with STIM1 floxed mice (STIM1f/f). Primary VSMC isolated from STIM1Δ/Δ SMC and the control STIM1f/f mice were used for in vitro characterization. VSMC was cultured in osteogenic media for 3 weeks; calcification was assessed by Alizarin red staining. Total calcium content was quantified by Arsenazo III method. MVs were collected by sequential centrifugation and characterized by electron microscopy. For in vivo characterization of vascular calcification, the mice were bred into the atherogenic ApoE−/− background.ResultsSTIM1 deletion in the STIM1Δ/Δ SMC VSMC promoted VSMC calcification, although STIM1 deficiency has been linked to decreased calcium signals in smooth muscle cells. Increased release of MVs was demonstrated with the STIM1Δ/Δ SMC VSMC compared with the control STIM1f/f VSMC. Using the atherogenic ApoE−/− model, we demonstrated that SMC‐specific STIM1 deficiency increased atherosclerotic vascular calcification in vivo. Consistently, increased MVs were determined in the serum of the STIM1Δ/Δ SMC mice. Mechanistically, we found that STIM1 deficiency did not affect VSMC proliferation and survival, but decreased the expression of SMC‐specific α‐actin. The size of MVs released from STIM1f/f and STIM1Δ/Δ SMC VSMC appeared similar. However, MVs from the STIM1Δ/Δ SMC VSMC contained a greater amount of calcium compared with those from the STIM1f/f VSMC. Furthermore, immunofluorescent staining identified a rearrangement of actin‐filament structure in the STIM1Δ/Δ VSMC, a critical cellular event that controls the release of MVs.ConclusionsThese studies have determined a new causative effect of VSMC‐expressed STIM1 on atherosclerotic vascular calcification; and identified a novel link connecting STIM1 and actin‐cytoskeleton rearrangement in regulating MV release and vascular calcification.Support or Funding InformationThis work was partially supported by grants from the National Institutes of Health HL092215, DK100847 and Veterans Administration BX001591 (project 2) to YC. YY was supported by American Heart Association Grant 14POST20450117.
111In‐DANBIRT as An in Vivo SPECT/CT Imaging Tool for The Expression of LFA‐1 in The Inflammatory Process of Atheroma Development
Authors: Mota Roberto Ivan , Daniels Tamara , Nysus Monique , Lucas Selita , Norenberg Jeffrey , Campen Matthew 
Published: November 2023

ObjectiveAssess inflammatory leukocyte presence and accumulation in vascular atherosclerotic plaque using 111In‐DANBIRT as a non‐invasive diagnostic imaging tool.Methods6 week ApoE KO mice were fed either normal or high fat chow (n=8 per group) for 8 weeks to induce vascular atherosclerotic lesions. SPECT/CT imaging was performed 3 hours post injection of ~700uCi of 111In‐DANBIRT at baseline, 4 weeks and 8 weeks. Whole body Autoradiography was performed 24 hours post injection after the 8 week time point. Image processing and analysis was performed by region of interest (ROI) determination in relationship to voxel and volume, uptake quantification was normalized to muscle uptake. Blood partition assays were conducted to ascertain specific binding components.ResultsAutoradiography and SPECT/CT analysis characterized 111In‐DANBIRT distribution in cardiovascular tissues (heart, aortic arch, descending aorta, carotids). A longitudinal increase in uptake was evidenced when comparing high fat diet to normal diet mice along our different time points. Increased uptake was evidenced in the thymus, which would be consistent with increased immune response to vascular injury. Whole blood isolation correlated these findings showing increased specific uptake by neutrophils in our ozone‐exposed group.ConclusionsThis pilot study showed that ApoE KO mice on Western diet exhibit increased cardiovascular and immune tissue uptake of 111In‐DANBIRT compared to mice on a normal diet. We still need to assess vascular lesion development in our disease model using biochemical markers and histology. Findings support future investigation of a more advanced vascular disease model that will validate 111In‐DANBIRT as a diagnostic tool for assessment of inflammation in cardiovascular injury models.Support or Funding InformationData was generated in the Keck‐UNM Small‐Animal Imaging Resource Shared Resource Center supported by the University of New Mexico Health Sciences Center, the UNM Cancer Center, and UNM College of Pharmacy. Grant numbers: NIH P30CA118100‐06 & ES0014639.
Sialylation and PECAM‐1‐Dependent Ligand Binding
Authors: Parambath Andrew , Abraham Valsamma , Hyun Sang Won , Liu Anguo , Goldblum Simeon E , DeLisser Horace 
Published: November 2023

PECAM‐1, a sialylated cell surface molecule expressed on endothelial cells (ECs), has been implicated in both EC motility and angiogenesis. Neuraminidase 1 (NEU1) sialidase inhibits wound‐induced EC migration and disrupts in vitro tube formation though PECAM‐1‐desialylation. The mechanisms by which desialylation of PECAM‐1 disrupts its activity are undefined. The effects of PECAM‐1 on cell function are mediated by the binding of PECAM‐1 to other PECAM‐1 molecules (homophilic) or to non‐PECAM‐1 molecules (heterophilic), such as heparin/heparan sulfate glycosaminoglycan (GAG)‐containing proteoglycans. We hypothesized that desialylation of PECAM‐1 disrupts PECAM‐1‐dependent ligand binding. For these studies, we employed REN cells (an EC surrogate) expressing mouse PECAM‐1 in mixed aggregation studies in which PECAM‐1‐expressing REN cells are mixed with control REN cells. In this assay, homophilic interactions result in aggregates that are enriched for PECAM‐1‐expressing cells, while heterophilic binding generates aggregates composed of both PECAM‐1 and non‐PECAM‐1 cell types. Analysis of 5‐cell aggregates in mixed aggregation studies for the PECAM‐1‐expressing REN cells resulted in comparable proportions of homophilic (46%) versus heterophilic (34%) aggregates. However, the formation of both homophilic and heterophilic aggregates was suppressed by adenoviral‐mediated overexpression of NEU1 in the PECAM‐1‐expressing REN cells compared to those that were transduced with adenovirus‐expressing GFP alone or when a catalytically‐inactive mutant of NEU1 was overexpressed. These data suggest that NEU1‐mediated inhibition of in vitro EC activity involves the disruption of PECAM‐1‐dependent ligand interactions.Support or Funding InformationVeterans Administration Merit Award
Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility
Authors: Ponce Alexander Garcia , Madrid Alí Francisco Citalán , Robles Hilda Vargas , Betanzos Abigail , Nava Porfirio , Zarbock Alexander , Rottner Klemens , Vestweber Dietmar , Schnoor Michael 
Published: November 2023

Inflammatory processes lead to changes in vascular permeability via the opening and closure of cell‐cell contacts. The actin cytoskeleton plays a crucial role in the regulation of endothelial cell contacts and thus permeability. Several molecules regulate actin cytoskeleton dynamics, one of these being cortactin, an actin nucleator protein capable of directly bind to Arp2/3 complex, filamentous‐(F)‐actin and additionally, to molecules involved in the regulation of actin dynamics such as myosin light chain kinase (MLKC) and ezrin, but also, proteins from cell contacts such as ZO‐1. These capabilities render cortactin as a potential regulator of endothelial cell actin dynamics and thus cell‐cell contacts. We already showed that cortactin regulates vascular permeability in vivo via Rap1, part of this regulation depends on Rap1 ability to regulate the actin cytoskeleton by blocking RhoA activity, a very well‐studied enhancer of vascular permeability. However, the exact mechanism that contributes to increased permeability without cortactin is not known. In a mass spectrometric analysis, we found that ROCK1 is induced ~3‐fold in cortactin‐KO endothelium. This was confirmed by Western blot and immunofluorescence of tissues from cortactin‐KO mice and human HMEC‐1 cells stably depleted for cortactin. Concomitantly, we observed increased phosphorylation of myosin light chain (MLC) and more stress fibres without cortactin. Other molecules involved in stress fibres formation and stabilization such as mDia1, MLCK and ezrin, were not affected by cortactin absence. Secretion of the endogenous hormone adrenomedullin (ADM), that is known to activate Rap1 and counteract formation of stress fibres, is reduced in cortactin‐KO mouse serum and supernatant of cortactin‐depleted HMEC‐1 cells. Importantly, adrenomedullin administration or ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling ctomyosin contractility with consequences for endothelial barrier integrity.Support or Funding InformationCONACyT grants 179895 and 233395 to MS and an international, bilateral grant of CONACyT and the German Ministry for Education and Research (BMBF); 207268/01DN14039 to MS and AZ)
Inhibiting Cardiac Fibrosis in Myocardial Infarction by CXCL10 Agonist Peptide
Authors: Espinoza Liliana , Jaynes Jesse , Bodnar Richard , Willis Monte S. , Yates Cecelia C. 
Published: November 2023

ObjectiveHeart failure is the leading cause of death and disability in the world, with 7.4 million deaths occurring in 2012 alone. Despite marked improvements in nearly every cardiovascular disease including heart attacks, heart failure has uniquely increased in prevalence and lacks therapeutic options targeting the progressive fibrotic response that commonly characterizes its pathogenesis. Much like the compensatory process seen in scarring of wounded skin, the heart responds to ischemia, hypertension, and common genetic mutations by undergoing extensive remodeling. However, overcompensation of the remodeling process often leads to fibrosis—an excessive accumulation of extracellular matrix (ECM) components. This accumulation is the results of activated fibroblasts that release collagen, fibronectin, and other ECM components. While a minimum of fibrosis is sometimes critical for structure (e.g. myocardial infarction), beyond this it has numerous negative consequences in the heart, including decreasing pump function and contributing to the development of cardiac arrhythmias. Outside of the heart, the beneficial role of the chemokine CXCL10 and its receptor CXCR3 in reducing fibrosis is well established. In the present study, we test the hypothesis that a newly developed CXCL10 agonist (IP10 peptide) similarly reduces fibrosis in the heart using a co‐culture model in vitro, and determine its relevance to MI in vivo.MethodCardiac tissue from mice challenged with permanent LAD ligation model (or sham controls) at 2 weeks post‐myocardial infarction were stained for CXCL10, its receptor CXCR3, and ECM components by immunohistochemistry to determine the temporal regulation of these components in vivo. In parallel, cardiomyocytes (CM), cardiac fibroblasts (CF), or CM/CF co‐cultures were challenged with transforming growth factor beta (TGF‐β) and/or treatment with CXCL10 or IP‐10 peptide. The in vitro cellular fibrotic response was then assessed for ECM expression, migration pattern, and apoptosis by immunocytochemistry (IHC) and immunoblot for specific components.ResultsIHC analysis of the heart two weeks post‐MI demonstrate a decrease in CXCL10, yet an increase in its receptor CXCR3 vs. sham controls. Treatment of CM/CF co‐culture with IP‐10 peptide in combination with TGF‐β resulted in reduced expression of α‐smooth muscle actin, collagen I, fibronectin, and tenascin C, compared to TGF‐β alone. These findings were not observed in single cells (CM or CF) cultures.ConclusionOur data suggests that chemokine CXCL10 plays an anti‐fibrotic role in remodeling, myocardial infarction‐induced cardiac tissue.
Genetic Inactivation of DNMT3b Protects Against Activation of Osteogenic Signaling and Slows Progression of Aortic Valve Stenosis in Mice
Authors: Sritharen Yoginee , Roos Carolyn M. , Nwachukwu Nneka , Kafa Rami , Hagler Michael A. , Verzosa Grace C. , Zhang Bin , Miller Jordan D. 
Published: November 2023

BackgroundWhile recent work from our laboratory showed that DNA (cytosine‐5)‐methyltransferase 3 beta (DNMT3b) is significantly increased in tissue from humans with calcific aortic valve stenosis and is associated with increases in global genomic methylation, it is unknown whether the net effects of these changes are protective or deleterious. The objective of this study was to test the hypothesis that genetic reduction of DNMT3b allows for increased anti‐osteogenic gene expression, and consequently results in suppression of osteogenic signaling and slower progression of valvular stenosis.MethodsWe used Ldlr−/−/ApoB100/100 (LA) mice that were wild‐type for DNMT3b (LA‐DNMT3b+/+) or deficient in DNMT3b (LA‐DNMT3b+/−) and fed a western diet for six months.ResultsUsing quantitative real‐time RT‐PCR, we confirmed that expression of DNMT3b was reduced by ~50% in valves from haploinsufficient mice. In line with our hypothesis, reductions in DNMT3b were associated with increases in expression of FABP4 (which opposes osteogenesis) and Smad6 (which opposes canonical BMP signaling). Similarly, expression of the osteogenic genes Msx2 and Spp1 were substantially reduced in DNMT3b‐deficient mice. Critically, aortic valve cusp separation distance was significantly greater in DNMT3b‐deficient mice, demonstrating slower progression of valvular dysfunction. No other gross abnormalities were noted in the mice.ConclusionsCollectively, these data suggest that selectively reducing DNMT3b activity may be a viable therapeutic approach to slowing progression of calcific aortic valve stenosis in humans.Support or Funding InformationGrant Funding Source: Supported by NHLBI
Effects of physical exercise and thyroid hormone on myosin heavy chain expression in doxorubicin‐induced cardiotoxicity in rats
Authors: Campos Erica C , Souza Fernanda R , Freitas Ana C S , Ramos Simone G , Resende Elmiro S 
Published: November 2023

The cardiomyocytes membrane integrity and the presence of structural proteins play an essential role for cellular function. The structural proteins that maintain the sarcolemma integrity has been associated with cardiotoxic effects of doxorubicin, as example, the loss of actin‐myosin interaction. Two therapeutic options could be used to minimize the deleterious effects of cardiotoxic effects in contractile apparatus: the use of thyroid hormone and exercise. For analysis of myosin expression the animals were divided into seven groups identified as: control (C), exercise control (EC), thyroid hormone control (CHT), doxorubicin control (CDX), hormone and doxorubicin (HTDX), exercise and doxorubicin (EDX) and the hormone plus exercise and doxorubicin (HTEDX). The animals received intraperitoneal injections of doxorubicin hydrochloride three times a week, for two weeks, reaching a cumulative dose of 7.5 mg / kg. To the CHT groups, and HTDX HTEDX hormone was administered daily for four weeks, by oro gastric probe. The dose was 10 mg / 100g body weight. CE groups, EDX and HTEDX performed exercise. The swimming exercise was used, and with additional cumulative loading up to 5% of body weight. The training was conducted for 4 weeks with three weekly sessions of swimming. The expression of myosin heavy chain evaluated by Western blotting showed increase in myosin expression in HTDX and HTEDX groups. The results suggest that exposure to higher doses of thyroid hormone significantly increases the expression of myosin both in the presence and absence of exercise. Additional effects of exercise can contribute to other favorable results in cardiovascular system.Support or Funding InformationCAPES
Neonatal and Juvenile Ocular Development in Sprague‐Dawley Rats: a Histomorphological Study
Authors: Vrolyk Vanessa , Apreutese Ancuta , Gordon Cedric , Forster Roy , Graham Andrew , Palate Bernard , Haruna Julius , Benoit‐Biancamano Marie‐Odile 
Published: November 2023

This study aimed at evaluating the histomorphological developmental features of the eye in Sprague‐Dawley rats, with focus on the postnatal and juvenile periods. Tissues were collected from 51 rats, from both sexes, aged 1 to 30 days old. As in other altricial species, the eyes of newborn rats are underdeveloped and the eyelids remain closed for approximately two weeks after birth. During the postnatal development, dramatic changes occurred within the retina, when programmed cell death and cell differentiation coexisted. A complete separation of the inner nuclear layer from outer nuclear layer was first observed close to the end of the first week of life, while the nerve fiber, ganglion cell and inner plexiform layers almost reached maturity concurrently with the opening of the eyes, around the end of the second week. At birth, apoptotic bodies were frequently seen within the undifferentiated nuclear layer and peaked at post‐natal day (PND) 8 within the inner nuclear layer. In newborn animals, mitoses formed a contiguous band of proliferating cells within the undifferentiated nuclear layer, adjacent to the photoreceptors layer. Perinatally, the corneal stroma and sclera exhibited high cellularity and became thinner and less cellular with maturity. The corneal epithelium consisted of a single layer of non‐stratified columnar cells during the first ten PNDs, and transformed into 4 to 5 cell layers of stratified squamous epithelium at about PND 14. The lens continuously developed by addition of new lens fibers from the lens epithelium. Between PND8 and PND17, a band‐like region of apoptotic bodies was seen centrally within the zone of nucleated lens fibers. We conclude that, during the first month of life, the rat's eyes undergo a rapid phase of growth and almost reach adult maturity at PND30. These observations will serve as an historical database, useful in pediatric ocular drug development.Support or Funding InformationMitacs