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Title: Promoting endothelialisation to reduce vascular injury following coronary angioplasty and stent implantation
Author: Gallogly, Susan
ISNI:       0000 0004 7655 2434
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2017
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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.
Supervisor: Mills, Nick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: novel cyclin-dependent kinase ; coronary endothelial outgrowth cells