Abstract
Purpose :
We have recently demonstrated that corneal plasmacytoid dendritic cells (pDCs) contribute to the preservation of corneal avascularity. The aim of this study is to analyze the secretion of angiostatic molecules by pDCs and their effect on vascular endothelial cell proliferation and differentiation in vitro.
Methods :
pDC-eGFPxRAGKO mice (in which only pDCs express GFP) were injected subcutaneously with B16 Flt3-L melanoma tumor cells. Splenic pDCs were isolated by sorting for GFP at day 10. To study endothelial cell proliferation and tube formation, pDCs were cultured in transwell inserts (TW) with human umbilical vein endothelial cells (HUVECs) with and without underlying Matrigel-coated well for 1 and 5 days, respectively. Real-time (RT) qPCR of pDCs for endostatin (ES), thrombospondin (TSP)-1 and -2, metalloproteinase inhibitor 3 (TIMP-3) and platelet factor 4 (PF-4) were performed. Proliferation using a hemocytometer and differentiation of HUVECs by formation of tube-like structures were evaluated after addition of blocking antibodies for TSP-1 or PF-4 or their isotype controls. The central area of each well was monitored and total length of all tubes measured using Angiogenesis Analyzer for ImageJ.
Results :
RT-qPCR of pDCs in TW showed increased mRNA expression from day 1 to day 5 of PF-4 (7.27 fold; p<0.05) and TSP-1 (2.74 fold; p<0.05). ES mRNA showed no significant change, while TSP-2 and TIMP-3 mRNA levels decreased, although only significantly for TIMP-3 (2 fold; p<0.001). pDCs decreased proliferation of HUVECs, but the addition of PF-4 blocking antibody abolished this effect completely (p<0.05). No significant difference to controls was noted for blocking antibody to TSP-1. Addition of pDCs to HUVECs on Matrigel inhibited formation of tube-like structures as compared to controls without pDCs. Addition of PF-4 blocking antibody ameliorated the effect of decreased tube formation by pDCs significantly (p<0.05), restoring total tube length to control levels. However, blocking antibody to TSP-1 showed no significant difference to controls.
Conclusions :
Our study demonstrates that the angiostatic effect of pDCs on the reduction of vascular endothelial cell proliferation and differentiation in vitro is mediated by PF-4. Cell-based therapy using pDCs may be considered as potential treatment option for corneal angiogenesis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.