Microarray analysis to identify molecular mechanisms of radiation-induced microvascular damage in normal tissues

Radiation-induced vascular injury can be a serious problem for cancer survivors. In capillary vessels, this manifests as telangiectasia, causing cosmetic problems when occurring in the skin and more serious problems, e.g. excessive bleeding requiring surgery, when occurring in rectal or bladder mucosa. In addition, thrombotic, inflammatory, and fibrogenic events play an important role in the development of late radiation injury in many tissues. However, the sequence of these events and the relationship between various mechanistic pathways is unclear. The purpose of this project is to identify genes that are differentially expressed in tissues with manifest vascular damage, with the ultimate goal of intervening in this process to block the progressive development of tissue injury.

Microarray experiments were performed using amplified RNA isolated from irradiated mouse kidney and rectum, and from sham-irradiated controls, at 10 and 20 weeks after treatment. Tissue samples were also taken for histologic evaluation of vascular damage at 10, 20, and 30 weeks after irradiation. Expression profiles for irradiated and sham-irradiated samples were compared, and differentially expressed genes were identified after normalization procedures, using information from straight color, color reverse, and self-self experiments. The extent of overlap in expression profiles for kidney and rectum during the phase of vascular damage was also examined.

The mouse kidney experiments showed upregulation of 31 genes at 10 weeks and 42 upregulated genes at 20 weeks. Only 20 genes showed significantly increased expression at both time points. Some of these genes were of particular interest in terms of their known involvement in vascular injury and signal transduction pathways. Irradiated mouse rectum had 278 upregulated genes at 10 weeks and 86 upregulated genes at 20 weeks. Only 19 of the genes upregulated during the period of identified telangiectasia (10-20 weeks) were common to both tissues. These included jagged 1 and Kruppel-like factor 5 (KLF5), which are reported to play a role in vascular development and remodeling.

Microarray analysis of RNA from irradiated normal tissues is an effective tool for identifying new genes of potential interest in the development of late tissue injury. Such experiments should be regarded as generating testable hypotheses for mechanisms of radiation-induced injury.