Korean J Ophthalmol.  2018 Feb;32(1):70-76. 10.3341/kjo.2016.0115.

Relationship between Pericytes and Endothelial Cells in Retinal Neovascularization: A Histological and Immunofluorescent Study of Retinal Angiogenesis

• Choi SH
• Chung M
• Park SW
• Jeon NL
• Kim JH
• Yu YS

• KMID: 2402723
• DOI: http://doi.org/10.3341/kjo.2016.0115

Abstract

PURPOSE
To evaluate the relationship between pericytes and endothelial cells in retinal neovascularization through histological and immunofluorescent studies.
METHODS
C57BL/6J mice were exposed to hyperoxia from postnatal day (P) 7 to P12 and were returned to room air at P12 to induce a model of oxygen-induced retinopathy (OIR). The cross sections of enucleated eyes were processed with hematoxylin and eosin. Immunofluorescent staining of pericytes, endothelial cells, and N-cadherin was performed. Microfluidic devices were fabricated out of polydimethylsiloxane using soft lithography and replica molding. Human retinal microvascular endothelial cells, human brain microvascular endothelial cells, human umbilical vein endothelial cells and human placenta pericyte were mixed and co-cultured.
RESULTS
Unlike the three-layered vascular plexus found in retinal angiogenesis of a normal mouse, angiogenesis in the OIR model is identified by the neovascular tuft extending into the vitreous. Neovascular tufts and the three-layered vascular plexus were both covered with pericytes in the OIR model. In this pathologic vascularization, N-cadherin, known to be crucial intercellular adhesion molecule, was also present. Further evaluation using the microfluidic in vitro model, successfully developed a microvascular network of endothelial cells covered with pericytes, mimicking normal retinal angiogenesis within 6 days.
CONCLUSIONS
Pericytes covering endothelial cells were observed not only in vasculature of normal retina but also pathologic neovascularization of OIR mouse at P17. Factors involved in the endothelial cell-pericyte interaction can be evaluated as an attractive novel treatment target. These future studies can be performed using microfluidic systems, which can shorten the study time and provide three-dimensional structural evaluation.

Keyword

Endothelial cells; Microfluidics; Oxygen induced retinopathy; Pericytes; Retinal neovascularization

MeSH Terms

Animals
Brain
Cadherins
Endothelial Cells*
Eosine Yellowish-(YS)
Fungi
Hematoxylin
Human Umbilical Vein Endothelial Cells
Humans
Hyperoxia
In Vitro Techniques
Lab-On-A-Chip Devices
Mice
Microfluidics
Microvessels
Neovascularization, Pathologic
Pericytes*
Placenta
Retina
Retinal Neovascularization*
Retinaldehyde*
Cadherins
Eosine Yellowish-(YS)
Hematoxylin
Retinaldehyde

Figure

• 

  Fig. 1 Normal development of retinal angiogenesis. (A) The retinas of normal mice at day (P) 4 to P26 were examined using H&E staining and were photographed under a microscope. (B) The retinas of normal mice at P4 to P26 were stained for endothelial cells with isolectin B4 (red) and for cell nuclei with DAPI (4′,6-diamidino-20 phenylindole, blue). G = ganglion cell layer; I = inner nuclear layer; O = outer nuclear layer; s = superficial plexus; i = intermediate plexus; d = deep plexus.

• 

  Fig. 2 Endothelial cell and pericyte interactions in normal mice and the oxygen-induced retinopathy (OIR) model. (A) Whole retina flat mount pictures of normal mice (left) and the OIR model (right) were stained for endothelial cells with isolectin B4 (red) (scale bar 1 mm). (B) The retinas of normal mice (left) and the OIR model (right) were stained for endothelial cells with isolectin B4 (red), and for pericytes with NG2 (green). In the OIR model, pericytes were found covering the neovascular tufts that extended into the vitreous (scale bar 20 µm). (C) The retinas of normal mice (left) and the OIR model (right) were stained for endothelial cells with isolectin B4 (green), for cell nuclei with 4′,6-diamidino-20 phenylindole (DAPI, blue) and for pericytes with NG2 (red). Endothelial cells and pericytes existed both in three-layered plexuses and neovascular tufts.

• 

  Fig. 3 N-cadherin expression in the oxygen-induced retinopathy model. The retinas of oxygen-induced retinopathy model mice were stained for endothelial cells with isolectin B4 (green), for cell nuclei with DAPI (4′,6-diamidino-20 phenylindole, blue), for pericytes with NG2 (red) and for N-cadherin (white, arrows).

• 

  Fig. 4 N-cadherin and pericyte expression in vascular networks using a microfluidic model. (A,B) Scheme of the microfluidic in vitro model that mimics retinal angiogenesis. (C) The angiogenesis model formed microvascular networks (red) was covered with pericytes (green) within 6 days. (D) Collagen IV (red) is deposited between the endothelial walls (white) and pericytes (green) at day 6. (E) N-cadherin (red) and α-smooth muscle actin (SMA, green) were co-expressed on pericytes covering the blood vessel (white) at day 6. HUVEC, human umbilical vein endothelial cell.

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