Development of in vitro models of native and injured vasculature is crucial for better understanding altered wound healing in disease, device implantation, or tissue engineering. Conditions were optimized using polyethyleneteraphalate transwell filters for human aortic endothelial cell (HAEC)/smooth muscle cell (HASMC) co-cultures with divergent HASMC phenotypes ('more or less secretory') while maintaining quiescent HAECs. Resulting HASMC phenotype was studied at 48 and 72 h following co-culture initiation, and compared to serum and growth factor starved monocultured 'forced contractile' HASMCs. Forced contractile HASMCs demonstrated organized alpha-smooth muscle actin filaments, minimal interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion, and low intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor expression. Organization of alpha-smooth muscle actin was lost in 'more secretory' HASMCs in co-culture with HAECs, and IL-8 and MCP-1 secretion, as well as ICAM-1, VCAM-1, and tissue factor expression were significantly upregulated at both time points. Alternately, 'less secretory' HASMCs in co-culture with HAECs showed similar characteristics to forced contractile HASMCs at the 48 h time point, while by the 72 h time point they behaved similarly to 'more secretory' HASMCs. These co-culture systems could be useful in better understanding vascular healing, however there remain time constraint considerations for maintaining culture integrity/cell phenotype.
Understanding endothelial cell (EC)/blood/biomaterial interactions is crucial for the advancement of cardiovascular devices that often fail because of the lack of nonthrombogenic biomaterials. To begin to assess these interactions, a static EC/blood cell/biomaterial model was used. Isolated blood cells were pretreated with model biomaterial beads with different surface chemistries: polystyrene (PS), and PS beads grafted with 3-kDa polyethylene glycol (PEG) with either a hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH(2)) terminal group at 5.4 or 54 x 10(4) beads/mL. Biomaterial-treated monocytes, neutrophils, or platelets were applied to human umbilical vein ECs (HUVECs) for 5 or 24 h of static coculture, and the resultant procoagulant HUVEC phenotype was characterized using several methods. Flow cytometry was used to assess surface expression of tissue factor (TF), adenosine triphosphate diphosphohydrolase, phosphatidylserine, and thrombomodulin, a functional TF assay was used to assess TF activity, and a plasma recalcification assay examined clotting times on HUVECs. Static coculture of HUVEC with biomaterial-treated neutrophils induced a procoagulant phenotype as exemplified by upregulation of TF expression and total functional activity, and downregulation of adenosine triphosphate diphosphohydrolase and thrombomodulin expression. The plasma recalcification assay demonstrated that HUVECs cocultured with biomaterial-treated monocytes significantly shortened clotting times, with some effect of similarly treated neutrophils.
Model in vitro culturing systems were developed to analyze roles of biomaterial-induced leukocyte activation on endothelial cell (EC) and smooth muscle cell (SMC) phenotype, and their crosstalk. Isolated monocytes or neutrophils were pretreated with model biomaterial beads and applied directly to "more secretory" (cultured in media containing 5% fetal bovine serum) or forced contractile (serum and growth factor starved) human aortic SMCs (HASMCs), or to the human aortic EC (HAEC) surface of HAEC/HASMC cocultures (HASMC phenotype varied to be "more or less secretory") for 5 or 24 h of static culture. Surface expression of proinflammatory [ICAM-1, VCAM-1, E-selectin], procoagulant (tissue factor), and anticoagulant (thrombomodulin) markers, as well as HAEC proliferation, were assessed by flow cytometry. Incubation of HAEC with biomaterial-pretreated monocytes (and neutrophils to lesser degree) suppressed HAEC proliferation and induced a proinflammatory/procoagulant HAEC phenotype. This HAEC phenotype was amplified in coculture with "more secretory" HASMCs and subdued in coculture with "less secretory" HASMCs. Direct incubation of biomaterial-pretreated monocytes or neutrophils with "more secretory" HASMCs further increased HASMC ICAM-1 and tissue factor expression. Direct incubation of biomaterial-pretreated monocytes or neutrophils with forced contractile HASMCs upregulated ICAM-1, VCAM-1, and tissue factor expression above the presence of serum-containing media alone.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.