Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in ...detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.
Purpose
Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial ...cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction.
Methods
In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo.
Results
CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo.
Conclusions
CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.
Rationale and Objectives: New therapeutic approaches that promote repair of the vasculature following injury are necessary to improve the clinical outcomes in patients undergoing angioplasty and ...stenting for coronary artery disease. I sought to establish a new cellular model to study the coronary endothelium in patients with acute myocardial infarction, and to utilise this model to identify novel polymer substrates that promote endothelialisation of coronary stents. Furthermore, I evaluated the potential of a novel cyclin-dependent kinase (CDK) inhibitor (AT7519) to resolve the acute neutrophil-mediated inflammatory response to stenting without adversely affecting the function and integrity of the endothelium. Methods: In 49 patients with acute myocardial infarction atherothrombotic specimens were isolated from the coronary circulation, dissected and cultured to obtain coronary endothelial outgrowth (CEO) cells. These cells were phenotyped, underwent assessments of proliferation and function (attachment, wound closure and tubule formation) in vitro and the capacity for angiogenesis in vivo was investigated. For comparison, endothelial cells from peripheral blood (late outgrowth endothelial cells EOCs) and human umbilical veins (HUVECs) were used. Polyacrylate (PA) and polyurethane (PU) polymers with high attachment of CEO cells but low attachment of neutrophils and monocytes were identified by high-throughput polymer microarrays. Polymers that facilitated CEO cell attachment under steady-state flow in an IBIDI chamber and supported endothelial cell proliferation were selected for further testing in a Badimon chamber to quantify thrombus formation as well as platelet and leucocyte attachment. Optimal concentrations of AT7519 to induce neutrophil apoptosis were assessed by morphological assessments of pyknosis. The effect of these concentrations on endothelial cell function and viability was assessed in vitro. Results: CEO was obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with a cobblestone morphology and high expression of CD146 (94±6%) and CD31 (87±14%). CEO cells had lower proliferative capacity to umbilical vein endothelial cells (23.0±6.8 versus 55.5±5.3 cumulative population doubling level, P < 0.001) but not other cells. CEO cells had a similar potential to control endothelial cells in other assays of endothelial cell function (cell attachment, migration and tubule formation, P > 0.05 for all) in vitro. Unlike HUVECs and EOCs, CEO cells did not incorporate into new host vessels in vivo. Polymer microarray libraries (337 polymers) identified PA309, an amine-functionalized methacrylate-based co-polymer, as having high endothelial cell attachment (~1,000 nuclei/mm2) with low inflammatory cell attachment ( < 200 nuclei/mm2). PA309 and PA318 supported endothelial cell attachment, migration and retention to the same extent as the collagen or 8G7 control (P > 0.05). PA309, but not PA319, supported CEO cell proliferation compared to collagen (3.5±1.3 versus 4.5±0.2 cumulative population doubling level CPDL). In ex vivo studies, PA309 (2,242±3,200μm2) and PA318 (3,043±4,054μm2) reduced thrombus formation compared to control (11,851±8,118μm2) (P < 0.01 and P < 0.001 respectively). Attachment of platelets and leucocytes was equivalent to conventional bare metal stents in clinical use. AT7519 at a concentration of 0.1μmοl/L induced neutrophil apoptosis compared to vehicle control (11±1 versus 1±1 nuclei pyknosis, P < 0.05) but did not inhibit CEO cell function (proliferation: 2.7±0.4 versus 2.8±0.5 fold-increase, attachment: 32±23 versus 57±5% attachment, wound closure: 94±8% versus 70±19%, tubule formation: 44±6 versus 58±11 tubule structures, P > 0.05 for all). Apoptosis and cytotoxicity was not observed in CEO cells at this concentration. Conclusions: Coronary endothelial outgrowth cells can be reliably isolated and cultured from thrombectomy specimens and represent a novel and relevant model to study endothelial cell function. Polymer PA309 promotes coronary endothelial cell attachment and expansion in vitro with similar potential to collagen and inhibits thrombus formation ex vivo. Furthermore, the CDK inhibitor AT7519 selectively induces neutrophil apoptosis with no adverse effect on endothelial cells at a defined concentration in vitro. Controlled elution of AT7519 and a PA309-coated coronary stent has major potential to promote the resolution of neutrophilic inflammation and promote endothelialisation, which could improve the clinical outcome for patients following coronary angioplasty and stenting for coronary artery disease.
Glutaredoxin (Grx)-catalyzed deglutathionylation of protein-glutathione mixed disulfides (protein-SSG) serves important roles in redox homeostasis and signal transduction, regulating diverse ...physiological and pathophysiological events. Mammalian cells have two Grx isoforms: Grx1, localized to the cytosol and mitochondrial intermembrane space, and Grx2, localized primarily to the mitochondrial matrix Pai, H. V., et al. (2007) Antioxid. Redox Signaling 9, 2027-2033. The catalytic behavior of Grx1 has been characterized extensively, whereas Grx2 catalysis is less well understood. We observed that human Grx1 and Grx2 exhibit key catalytic similarities, including selectivity for protein-SSG substrates and a nucleophilic, double-displacement, monothiol mechanism exhibiting a strong commitment to catalysis. A key distinction between Grx1- and Grx2-mediated deglutathionylation is decreased catalytic efficiency ( k cat/ K M) of Grx2 for protein deglutathionylation (due primarily to a decreased k cat), reflecting a higher p K a of its catalytic cysteine, as well as a decreased enhancement of nucleophilicity of the second substrate, GSH. As documented previously for hGrx1 Starke, D. W., et al. (2003) J. Biol. Chem. 278, 14607-14613, hGrx2 catalyzes glutathione-thiyl radical (GS (*)) scavenging, and it also mediates GS transfer (protein S-glutathionylation) reactions, where GS (*) serves as a superior glutathionyl donor substrate for formation of GAPDH-SSG, compared to GSNO and GSSG. In contrast to its lower k cat for deglutathionylation reactions, Grx2 promotes GS-transfer to the model protein substrate GAPDH at rates equivalent to those of Grx1. Estimation of Grx1 and Grx2 concentrations within mitochondria predicts comparable deglutathionylation activities within the mitochondrial subcompartments, suggesting localized regulatory functions for both isozymes.
Quality of care of patients with acute myocardial infarction (AMI) has received intense attention. However, it is unknown if a structured initiative for improving care of patients with AMI can be ...effectively implemented at a wide variety of hospitals.
To measure the effects of a quality improvement project on adherence to evidence-based therapies for patients with AMI.
The Guidelines Applied in Practice (GAP) quality improvement project, which consisted of baseline measurement, implementation of improvement strategies, and remeasurement, in 10 acute-care hospitals in southeast Michigan.
A random sample of Medicare and non-Medicare patients at baseline (July 1998--June 1999; n = 735) and following intervention (September 1--December 15, 2000; n = 914) admitted at the 10 study centers for treatment of confirmed AMI. A random sample of Medicare patients at baseline (January--December 1998; n = 513) and at remeasurement (March--August 2001; n = 388) admitted to 11 hospitals that volunteered, but were not selected, served as a control group.
The GAP project consisted of a kickoff presentation; creation of customized, guideline-oriented tools designed to facilitate adherence to key quality indicators; identification and assignment of local physician and nurse opinion leaders; grand rounds site visits; and premeasurement and postmeasurement of quality indicators.
Differences in adherence to quality indicators (use of aspirin, beta-blockers, and angiotensin-converting enzyme ACE inhibitors at discharge; time to reperfusion; smoking cessation and diet counseling; and cholesterol assessment and treatment) in ideal patients, compared between baseline and postintervention samples and among Medicare patients in GAP hospitals and the control group.
Increases in adherence to key treatments were seen in the administration of aspirin (81% vs 87%; P =.02) and beta-blockers (65% vs 74%; P =.04) on admission and use of aspirin (84% vs 92%; P =.002) and smoking cessation counseling (53% vs 65%; P =.02) at discharge. For most of the other indicators, nonsignificant but favorable trends toward improvement in adherence to treatment goals were observed. Compared with the control group, Medicare patients in GAP hospitals showed a significant increase in the use of aspirin at discharge (5% vs 10%; P<.001). Use of aspirin on admission, ACE inhibitors at discharge, and documentation of smoking cessation also showed a trend for greater improvement among GAP hospitals compared with control hospitals, although none of these were statistically significant. Evidence of tool use noted during chart review was associated with a very high level of adherence to most quality indicators.
Implementation of guideline-based tools for AMI may facilitate quality improvement among a variety of institutions, patients, and caregivers. This initial project provides a foundation for future initiatives aimed at quality improvement.
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