Inducible Overexpression of Endothelial ProNGF as A Mouse Model to Study Retinal Microvascular Dysfunction

Abstract

Purpose: Purpose: Diabetic retinopathy is a progressive disease characterized by early neuro-and vascular degeneration leading to a later blinding proliferative phase. Previously, our lab has demonstrated involvement of the nerve growth factor precursor (proNGF) and its receptor p75^NTR in diabetes-induced retinal glial inflammation, vascular permeability and acellular capillary formation using the streptzotocin model of diabetes. The aim of this study is to dissect the autocrine action of proNGF in endothelial cells apart from the paracrine effects from proNGF on glia.

Methods: Using Cre-Lox technology, we have developed an inducible endothelial-specific proNGF transgenic mouse. A construct of cleavage-resistant proNGF123,originally obtained from Dr. Kenneth Neet (University of Chicago), was engineered to produce GFP-conjugated-proNGF construct and proNGF^Loxp mice. Transgenic proNGF^Loxp mice were backcrossed with C57BL6J mice from Jackson Laboratories for 5-generations before actual experiments. Cre-VE-Cadherin mice were a kind gift of Dr. Luisa Iruela-Arispe (UCLA). Age matched groups of Cre or Cre-proNGF^Loxp mice received five-consecutive intraperitoneal injections of tamoxifen in sunflower oil to activate proNGF expression. Retinal vascular permeability was assessed by extravasation FITC-dextran (70,000 daltons).

Results: Protein expression was evaluated by Western blot and immunohistochemistry. Results: There was no significant difference in the weight or blood glucose levels of Cre-proNGF^Loxp mice with their Cre counterparts. Expression of Cre, GFP, and proNGF were confirmed by Western blot. Examination of retinal lysates of Cre-proNGF^Loxp mice showed increased vascular permeability (1.8-fold) that was associated with marked expression of vascular GFP in Cre-proNGF^Loxp mice compared to Cre-controls.

Conclusions: The use of the Cre-Lox technology successfully generated viable proNGF^Loxp mice in contrast to the embryonic lethal systemic proNGF transgenic mouse. Understanding how endothelial specific proNGF contributes to retinal microvascular abnormalities independent of the diabetic milieu and interacting retinal cell types will provide valuable insights on mechanisms leading to diabetic retinopathy.