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

Keywords

ionizing radiation, adhesion molecules, endothelium, inflammatory response, late effects

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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