Neutralization of Autocrine TGF-B Results in Upregulation of CXCR4 in Haematopoietic Stem Cells (HSC) in vitro and Reduction in Volume of Choroidal Neovascular Lesions (CNV) in vivo

Abstract

Purpose:: TGFß is a pleiotropic regulator of HSC and hematopoiesis. HSC, known for their plasticity and repair function can differentiate into many cell types including RPE and endothelial cells. An understanding of HSC involvement in CNV development is incomplete. To increase our knowledge of HSC function in CNV, we asked whether TGF-ß1 blockade could lead to enhanced reparative potential by increasing HSC proliferation, survival, and expression for CXCR4, the receptor of the stem cell homing factor SDF-1. We also investigated whether increasing their reparative function by TGF-ß1 blockade had a beneficial or detrimental effect on CNV lesion size.

Methods:: HSC were treated with either TGF-ß antisense, scrambled oligonucleotide control or saline control. CXCR4 expression was examined by immunolabelling and FACS analysis. Mice underwent bone marrow (BM) irradiation and were transplanted with HSC pretreated with either TGF-ß antisense, scrambled oligonucleotide control or saline control and engraftment was examined (n=10). A second group underwent the laser rupture of Bruch’s membrane CNV model and were immediately injected with intravitreal saline, scrambled oligonucleotide, or TGF-ß morpholino antisense. A third group were injected systemically with HSCs pretreated with saline, scrambled oligonucleotide or TGF-ß morpholino antisense and then underwent induction of CNV (n=6 per group and condition). Lesion components and volume were quantified at 1 and 2 weeks post laser injury using flatmounts of the posterior poles imaged by laser scanning confocal microscopy.

Results:: Neutralizing autocrine TGF-ß1 resulted in upregulation of CXCR4 on HSC. Neutralization of TGF-ß1 in HSC resulted in a dramatic acceleration of the repopulation potential of the transplanted HSC and increasing homing to the BM and BM engraftment. Intravitreal injection of TGF-ß morpholino antisense resulted in a significant (P<0.05) decrease in lesion size compared to injection of scrambled antisense or saline controls.

Conclusions:: Blocking autocrine TGF-ß production induces upregulation of CXCR4 in HSCs, dramatically improves HSC function, and results in a decrease in CNV lesion size. Modulating TGFß-1 in HSC may represent a novel therapeutic strategy in the management of CNV.

Keywords: choroid: neovascularization • wound healing • cytokines/chemokines