Date

2015

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Bioengineering

First Adviser

Liu, Yaling

Other advisers/committee members

Lowe-Krentz, Linda; Ou-Yang, Daniel; Cheng, Xuanhong

Abstract

Endothelial cells form the inner lining of blood vessels and regulate key blood vessel functions including host defense reactions, vascular smooth muscle tone, angiogenesis, and tissue fluid homeostasis. The occurrence of a pathological condition can lead to inflammation, a protective and tightly regulated response involving host cells, blood vessels and proteins. This process is promoted by circulating cytokines and other chemical mediators such as tumor necrosis factor-alpha (TNF-α), interleukins, thrombin, a few examples. Inflammation can be acute or chronic in nature and is characterized by specific cell receptor expression patterns on the endothelial layer and an increase in endothelial cell-cell gaps. Upregulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial surface occurs during inflammation, and ICAM-1 plays an important role in leukocyte adhesion and recruitment. Understanding the dynamic nature of receptor expression and endothelial cell gap formation during inflammation would provide fundamental physiological information and is also essential for evaluating drug and other biomolecules targeted binding and uptake by endothelial cells.The far reaching accessibility combined with heterogenic behaviour make microvascular endothelium an attractive target for targeted drug delivery. Dynamic and complex processes governing the targeted drug particle binding and distribution on blood microvasculature are still partially understood. Part of this work focuses on the characterization of particle delivery in microcirculation on an ICAM-1 coating based blood vessel mimicking microfluidic device. In microvasculature the vessel size is comparable to that of red blood cells (RBCs) and the existence of blood cells largely influences the dispersion and binding distribution of drug loaded particles. Various factors that influence particle distribution and delivery such as the vessel geometry, shear rate, blood cells, particle size, particle antibody density were considered in this study. Better understanding of the pathologically challenged local endothelial cell layer microenvironment can help us engineer drug carriers decorated with specific biomolecules which can improve the pharmacokinetics and pharmacodynamics of drugs. In this study we also developed a biomimetic blood vessel model by culturing confluent, flow aligned, Endothelial cells in a microfluidic platform, capable of being treated with inflammatory mediators locally. Primary bovine aortic endothelial cells (BAOECs) were grown on semi-permeable membrane with pores that separates an upper and lower channel made of polydimethylsiloxane (PDMS). This dual channel design allowed localized direct TNF-α treatment on the endothelial cell layer leading to expression of surface ICAM-1. This model simulated spatially controlled healthy and pathologically challenged endothelial cells in the same channel and thus has the ability to investigate the microenvironment of locally activated endothelial cells. We characterized endothelial cell culture in this platform and and performed real-time in situ characterization of localized pro-inflammatory endothelial activation. Anti-ICAM-1 coated particles (210 nm and 1 µm diameter) of different antibody coating densities were used as imaging probes and availability of ICAM-1 was probed. This allowed the investigation of spatial resolution and accessibility of ICAM-1 molecules on endothelial cells for targeted binding studies. Anti-ICAM-1 coated particles specifically bound to TNF-α activated BAOECs in an antibody coating density, FSS and particle size dependent manner. F-actin remodelling was also observed in TNF-α treated and downstream sections of the channel. This work has developed a more realistic in vitro vascular model that can independently integrate various factors to effectively mimic a complex physiological endothelial cell microenvironment and has been applied to study endothelial cell microenvironment under localized inflammatory triggering. Inflammatory responses in endothelial cells are characterized by an increase in vascular permeability by formation of intercellular gaps. The biomimetic blood vessel model developed by culturing confluent, flow aligned, and endothelial cells in a microfluidic platform (described prior) was utilized in characterizing the dynamic nature of vascular permeability under inflammation. BAOECs were subjected to in vivo levels of prolonged flow and then treated with thrombin, a serine protease. Thrombin induced profound increase of endothelial cell monolayer permeability in a rapid and reversible way. Endothelial cells cultured in the upper channel were exposed to media mixed with thrombin and a tracer molecule. Tracer molecule samples were collected real time and analysed using spectroscopy, and the dynamic nature of the process was studied. The remodelling of F-actin in BAOECs after thrombin treatment was also characterized for different time points. Better understanding of the dynamics involved in increased vascular permeability can help engineer strategies to enhance targeted drug delivery through these para-cellular gaps.

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