Abstract
Purpose:
Perivascular supporting cells including pericytes (PC) play an integral role during angiogenesis and control vascular remodeling, maturation, and stabilization of neoteric vessels. We recently showed that a Cyp1b1-deficiency in mice results in the attenuation of angiogenesis in vivo and the pro-angiogenic activity of endothelial cells (EC) in vitro. However, the contribution of PC, and more specifically the cell autonomous effects of Cyp1b1 in these processes, needs further investigation.
Methods:
Retinal PC were isolated form Cyp1b1+/+ and Cyp1b1-/- Immorto mice as previously described by us. The expression of Cyp1B1 was confirmed by Western blotting of cell lysates. Expression of PC-specific markers, integrins, and other cell adhesion molecules was evaluated by FACS analysis. Cell proliferation was assessed by counting the number of cells and Edu-Click-It DNA labeling. Apoptosis was determined by measuring caspase activation using Caspase-Glo 3/7-assay. The levels of oxidative stress were determined by staining cells with Dihydroethidium. Cell migration was assessed using scratch wound and transwell migration assays. VEGF production was determined using a mouse VEGF Immunoassay.
Results:
Retinal PC constitutively expressed Cyp1B1. Lack of Cyp1B1 was associated with enhanced proliferation, and decreased apoptosis of retinal PC. Mechanistically, the lack of Cyp1B1 was associated with increased oxidative stress and sustained NF-κB activation, which was reversed by the anti-oxidant N-acetylcysteine. These changes were concomitant with alterations in PC migration, adhesion, and expression of various extracellular matrix proteins including thrombospondin-2 (TSP2). Cyp1B1-deficient PC also expressed decreased levels of VEGF.
Conclusions:
Together our results suggest an important role for Cyp1B1 expression in the regulation of PC proliferation, migration, and survival through modulation of the intracellular oxidative state and NF-κB expression and/or activity. Thus, a lack of Cyp1B1 in PC may have a significant role in vascular dysfunction and integrity, contributing to the attenuation of angiogenesis.
Keywords: 700 retinal neovascularization •
634 oxidation/oxidative or free radical damage •
715 signal transduction: pharmacology/physiology