Early molecular changes in irradiated aortic endothelium

Irradiated aortic endothelial cells (EC) exhibit distinct morphological, fu nctional, and physiological responses to ionizing radiation (IR). However, the molecular basis for these responses has not been fully characterized. C ultured bovine and rat aortic endothelial cells were exposed to single trac tion doses (0-30 Gy) of gamma radiation. IR caused dose-dependent DNA stran d breaks which were repaired to near baseline levels within 30 min. A dose- dependent inhibition of cell growth was noted for IR greater than 1 Gy. At doses greater than 2.5 Gy, morphologic changes consistent with apoptosis an d loss of cell viability were present beginning 12-16 h after radiation, wi th subsequent detachment of EC from the cell monolayer. By Western blot ana lysis, expression of p53, gadd45, p21, and bar protein increased in a time- and dose-dependent manner; p53 expression was maximal at 3 h after IR, and gadd45, bar and p21 levels peaked at G h. By Reverse Transcriptase Polymera se Chain Reaction (RT-PCR), levels of p53 mRNA were not significantly incre ased after IR, whereas gadd45 exhibited time- and dose-dependent increase i n mRNA synthesis after IR. Activation of intracellular caspases, manifest b y proteolytic poly (ADP-ribose) polymerase (PARP) and lamin B cleavage, was maximal at 15 h after IR, concident with other indices of EC apoptosis, in cluding oligonucleosomal DNA degradation, TUNEL immunostaining, and morphol ogic changes. The tripeptide protease inhibitor z-Val-Ala-Asp (zVAD) preven ted PARP and lamin cleavage, DNA fragmentation, morphological changes, and cell detachment in irradiated EC. The combined data suggested that gamma ra diation induces a dose- and time-dependent sequence of early events in cult ured EC with modulate growth arrest, apoptosis, and possibly premature sene scence in surviving cells. (C) 2001 Wiley-Liss, Int.