Hypothesis / aims of study
Radiation cystitis (RC), a long-term side effect from radiation therapy for pelvic cancers, is characterized by inflammation, fibrosis and vascular damage. In addition to lower urinary tract symptoms, patients with RC suffer from hematuria, which can range from microscopic hematuria to gross hematuria with blood clots. In patients with hemorrhagic (radiation) cystitis, arresting bleeding is the primary focus. Choice of treatment is dependent on the severity of hematuria and includes bladder irrigation, instillation of astringent agents, hyperbaric oxygen therapy, and as a last resort formalin instillation and cystectomy [1]. However, existing treatments have high recurrence rates or can have severe side effects. The lack of mechanistic insight in the vascular defect of RC has limited the development of effective therapies. We hypothesize that radiation-induced damage to the vasculature drives the chronic inflammation, fibrosis and hemorrhaging in radiation cystitis patients. In this study we evaluated the effect of radiation damage on endothelial cell function and assessed the expression of ICAM-1. ICAM-1 is a ligand involved in the recruitment of leukocytes from the bloodstream, hereby supporting a pro-inflammatory phenotype.
Study design, materials and methods
For in vitro studies, primary human umbilical vein endothelial cells (HUVEC) were exposed to low (1 Gy) or high (6 Gy) dose irradiation. Cell proliferation, migration and cellular senescence were assessed 1, 3 and 7 days post-irradiation. At those same time points, the expression of ICAM-1 and VCAM-1 were determined using quantitative PCR. The bladder vasculature was subsequently studied in our radiation cystitis mouse model. The bladders of C57BL/6 mice received a single dose of 40 Gy irradiation using SARRP. Mice were monitored for urinary frequency and hematuria. Bladders were harvested at various time points after radiation exposure (from 2 days up to 1 year after radiation), processed for histology and analyzed for vascular health, fibrosis and inflammation. The expression of ICAM-1 in bladder vasculature was determined using RNAscope.
Interpretation of results
Our results indicate that radiation of primary endothelial cells disrupts normal endothelial function as measured by reduced proliferation and migration of HUVEC. A plausible mechanism is that radiation accelerates endothelial cell senescence, as we observed a higher number of senescent cells after irradiation without observing a change in the number of dead cells after radiation exposure. We detected increased ICAM-1 expression up to 7 days after irradiation. A previous study demonstrated that increased ICAM-1 expression is associated with senescence in endothelial cells [2], which supports our findings. In vivo, radiation results in visible damage to bladder vasculature, which include loss of vascularization, telangiectasia and hemorrhaging. These damaged blood vessels have higher levels of ICAM-1 expression 48 hours after radiation exposure, possibly due to irradiation-induced endothelial cell senescence. Ongoing studies are assessing ICAM-1 expression at later time points after irradiation in vivo and are determining if radiation-induced ICAM-1 expression results in increased leukocyte recruitment and adhesion.