Abstract
Purpose: :
Previous studies have shown that topical application of PEDF and docosahexaenoic acid (DHA) increases nerve area after lamellar keratectomy in rabbits (ARVO 2008). PEDF is a neurotrophic factor that stimulates the synthesis of neuroprotectin D1 (NPD1) in retinal pigment epithelial cells (Proc Natl Acad Sci U S A, 104(32):13158-63, 2007). Injury to the cornea promotes the synthesis of several growth factors important in wound healing. We investigated whether injury to the cornea promotes the synthesis and secretion of PEDF and if PEDF affects the expression of 15-lipoxygenease-1 (15-LOX1), the enzyme responsible for catalyzing the synthesis of NPD1.
Methods: :
Rabbit corneas in organ culture were injured (7mm epithelial debridement) and cultured for 12, 24 and 36 h. Total RNA was extracted from the epithelium and gene expression of PEDF was analyzed by RT-PCR. Forty eight hours after injury, epithelial cells and media were collected and analyzed by Western blot using a goat polyclonal IgG-PEDF antibody. Tissue was fixed and analyzed by immunofluorescence. In other experiments, human cornea epithelial cells (HCEC) were stimulated with 40ng/ml PEDF for 24 and 48 h, and expression of 15-LOX1 was measured by Western blot with a mouse monoclonal antibody.
Results: :
Significant increase in PEDF gene expression was found at 12 and 24 h after the wound in corneal epithelial cells. There were increased levels of PEDF in the media after 48 h of injury (p<0.01), while the expression of PEDF in the injured corneal epithelial cells was decreased (p<0.03). Stimulation of corneal epithelial cells with PEDF produced a 1.5- to 2.0-times increase in the expression of 15-LOX1 at 24 h and at 48 h, respectively.
Conclusions: :
Corneal injury promotes synthesis and secretion of PEDF from the epithelium. PEDF also increases the expression of 15-LOX1, which can then activate the synthesis of NPD1. Our results unveil new mechanisms of PEDF action that could be important in nerve regeneration and corneal epithelial integrity.
Keywords: growth factors/growth factor receptors • lipids • innervation: sensation