Measurement of Endothelial Cell Proliferation Rate in vivo Using 2H2O Labeling: A Kinetics Biomaker of Angiogenesis

An accurate method for measuring angiogenesis rate in tissues remains a high priority. We recently developed a stable isotope‐mass spectrometric technique for measuring cell proliferation rates in vivo by use of heavy water (2H 2 O) labeling. Here, we apply this technique to the measurement of endothelial cell (EC) proliferation rate and, thus, angiogenesis. EC were isolated from tissues by cell surface marker labeling and flow‐cytometry, using anti‐CD31 antibody and isolectin. Incorporation of deuterium into the deoxyribose moiety of EC DNA was measured by GC/MS. Under basal conditions, the replacement rate of renal EC was about 4.9% per week and for hepatic EC was about 10% per week. Whole EC isolated from xenograft tumor tissue in Nu‐/Nu‐ mice was 53%/wk. Twice weekly treatment with anti‐VEGF antibody (Avastin®) significantly reduced proliferation of normal renal and hepatic EC as well as xenograft tumor EC (10% with 2mg/kg and 20% with 20mg/kg). In some settings (2mg/kg anti‐VEGF), lower EC proliferation rate was not accompanied by reduced tumor cell proliferation rate. In contrast, gemcitabine, an approved tumor chemotherapeutic drug, exhibited broader anti‐proliferative activity compared to specific angiogenesis agents. In summary, a non‐radioactive technique has been developed for measuring rates of angiogenesis in tissues. This approach has potential applications in pathophysiology research and drug development.