R-Cadherin Blocking Peptides Disrupt Vascular Patterning and Targeting of Bone-Marrow Derived Hematopoeitic Stem Cells in the Developing Mouse Retinal Vasculature

Abstract

Abstract: : Purpose: Recently, a subset of hematopoietic stem cells (HSCs) have been shown to target and become incorporated into the developing mouse retinal vasculature. Additionally, the use of R-cadherin specific blocking antibodies has shown the importance of R-cadherin guidance cues in the formation of the retinal vascular layers. We have synthesized novel R-cadherin function-blocking peptides in order to assess the potential role of R-cadherin in normally developing retinal vasculature as well as the targeting of HSCs during retinal angiogenesis. Methods: Mutational analysis, structural information of similar cadherin family members, and sequence homology were used to generate novel peptides that potentially block trans-dimerization of mouse R-cadherin. The peptides were characterized in vitro by their ability to block R-cadherin mediated cell aggregation, and in vivo by the ability to disrupt normal developmental retinal vascularization. HSCs were preincubated with blocking R-cadherin antibodies or peptides prior to injection; confocal microscopy was used to assess targeting and incorporation of these and control-treated HSCs into the developing vasculature. Results: Cyclic and linear peptides directed against two separate regions of R-cadherin extracellular domain 1 specifically blocked R-cadherin, but not the closely related N-cadherin, mediated cell aggregation. These peptides also disrupted vascular patterning in vivo. In addition, blocking of R-cadherin on HSCs with these peptides or R-cadherin blocking antibodies effectively caused a large portion of injected HSCs to become mistargeted. Conclusions: We have identified regions of R-cadherin that mediate specific trans-dimerization and are important for endothelial cell guidance during normal retina development. In addition, our results suggest that R-cadherin is important for guidance and selective targeting of HSCs to specific sites of developing retinal vasculature.

Keywords: retinal neovascularization • retinal development • cell adhesions/cell junctions