Date of Award

6-2000

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Biomedical Engineering

Research Advisor

Mohammad F. Kiani

Committee

Douglas J. Goetz Thomas E. Merchant

Abstract

Ionizing radiation is a major therapy for cancer. Damage to the endothelium and upregulation of inflammatory responses are major side effects of radiotherapy, but the mechanisms behind these processes are not clear. A key component of the inflammatory response is leukocytes adhesion to the endothelium in a multi-step process involving leukocyte tethering to the endothelium, followed by rolling, firm adhesion/spreading and extravasation. Adhesion molecules mediate this process, particularly, E-selectin and ICAM-1, which are inducible adhesion molecules. E-selectin is involved in the initial tethering and rolling steps of the adhesion cascade while ICAM-1 appears to be involved in the latter steps. We have hypothesized that in response to irradiation, the endothelium becomes activated, increases its expression of adhesion molecules such as E-selectin and ICAM-1, and supports the increased interaction of leukocytes with the endothelium. To probe this hypothesis, we have investigated the expression of E-selectin and ICAM-1 on Human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HDMECs) and transformed microvascular endothelial cells (HMEC-1) at 5 hr, 24 hr, 48 hr and 72 hr post-irradiation. We have also developed an in vitro flow model to study the functional consequences of the radiation induced upregulation of adhesion molecules, primarily E-selectin and ICAM-1. Endothelial cells were grown in 35mm dishes and irradiated with a single dose of 10 Gy. HL60 (human promyelocytic leukemia) cells were perfused over the irradiated (IR) endothelial cells in a parallel plate flow chamber at shear stress of 0.5-2.0 dynes/cm 2 . Flow cytometry analysis at 5 hr, 24hr, 48 hr and 72 hr post-irradiation showed upregulation of E-selectin at 5 hr post-IR only on HDMECs. ICAM-1 was upregulated at 24 hr (HDMECs), and 48 hr (HUVECs and HMEC-1).
Flow assays revealed an increase in the rolling and adhesion of HL60 cells on 5 hr post-IR HDMEC. No rolling was observed on HUVECs and HMEC-1 monolayers 5 hr post-IR consistent with the absence of E-selectin from the flow cytometry data. Semi-static assays at 48 hr post-IR on HUVECs showed an increase in the number of adherent cells. There was a radiation dose dependent (5 Gy vs. 10 Gy) increase in the expression of adhesion molecules, which was significant at 72 hr post-IR. Our findings suggest that irradiation selectively impacts the functional aspects of the inflammatory response.

DOI

10.21007/etd.cghs.2000.0253

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Therapeutics Commons

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