The Influence of C-Ions and X-rays on Human Umbilical Vein Endothelial Cells

Abstract

Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells as a model system to examine the effect of low- and high-linear energy transfer (LET) radiation. Cells were exposed to 250 kV X-rays or carbon ions (C-ions) with the energies of either 9.8 MeV/u (LET = 170 keV/μm) or 91 MeV/u (LET = 28 keV/μm). Subculture of cells was performed regularly up to 46 days (~22 population doublings) post-irradiation. Immediately after exposure, cells were seeded for the colony forming assay. Additionally, at regular intervals, mitochondrial membrane potential (MMP) (JC-1 staining) and cellular senescence (senescence-associated β-galactosidase staining) were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay) as observed in other cell systems. For 9.8 and 91 MeV/u C-ions, relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time points, the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the MMP was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time point investigated. Altogether, our data indicate that shortly after exposure C-ions were more effective in damaging endothelial cells than X-rays. However, late damage to endothelial cells was not found for the applied conditions and endpoints.

Keywords: carbon ion therapy; carbon ions; cardiovascular disease; chromosome 13; endothelial cells; high-LET radiation; micronucleus formation; senescence-associated β-galactosidase.

Figure 1

Clonogenic cell survival of HUVEC. Cells were plated immediately after exposure to X-rays or C-ions. Data points represent the mean X ± SD from replicates stemming from one (C-ions) or three (X-rays) experiments. Curves were fitted by a linear function. Based on the α-values, clonogenic cell survival was found significantly (p < 0.01, Student’s t-test) different for the three radiation types.

Figure 2

Micronuclei formation 24 h after exposure. Following irradiation, cells were incubated with cytochalasin-B, and the amount of binucleated cells containing micronuclei was determined. Data points represent the mean X ± SEM (for data points with n = 2) or error was calculated according to Poisson statistics for data points stemming from one experiment. Curves for X-rays and 9.8 MeV/u C-ions were fitted as described. For 91 MeV/u C-ions, lines are drawn to guide the eye. Statistical analysis using a Student’s t-test revealed significant differences for 9.8 MeV/u C-ions when compared to X-rays (p < 0.01).

Figure 3

Apoptosis 48 h after exposure. Cells were fixed 48 h following radiation exposure to X-rays (A), 9.8 MeV/u C-ions (B), and 91 MeV/u C-ions (C). The fraction of apoptotic cells was determined according to morphological criteria of the nucleus. Error was calculated according to Poisson statistics. Fisher’s exact test revealed no significant differences between all samples (n = 1, p > 0.01).

Figure 4

Micronuclei formation in cells after extended culture time. Cells were fixed at several time points after exposure (w/o cytochalasin-B), and all cells harboring micronuclei were scored. The error was calculated according to Poisson statistics, and a Fisher’s exact test was performed (n = 1). Only 2 days after exposure, micronuclei formation was found significantly higher compared to the control (mean ± SD). Note that for better visualization, the samples 2 days after exposure to the different radiation types were plotted separated from each other despite stemming from the same time point.

Figure 5

Analysis of structural chromosome aberrations in HUVEC cultures (non-irradiated and irradiated) by means of the mFISH technique. The fractions of normal and aberrant diploid/hypodiploid cells (referred to as ~2N) and tetraploid/hypotetraploid cells (referred to as ~4N) are given (n = 1). The terms hypodiploidy or tetradiploidy indicate the loss of one or two chromosomes. Cells were analyzed in controls at a CPD level of 13 ± 2 and about 9 population doublings after exposure (CPD ~22).

Figure 6

Typical aberrations detected in HUVEC by means of the mFISH technique. (A) Hypotetraploid cell, one copy of chromosome 13 is lost. (B) Diploid cell, one chromosome is truncated (here: non-irradiated cells, CPD ~22).