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

Affiliations
  • 1Department of Ophthalmology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea. ysyu@snu.ac.kr
  • 2Mechanical Engineering, Seoul National University, Seoul, Korea.
  • 3Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea.
  • 4FARB (Fight against Angiogenesis-Related Blindness) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.

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.


Reference

1. Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med. 2011; 17:1359–1370.
Article
2. Yoshida A, Yoshida S, Ishibashi T, Inomata H. Intraocular neovascularization. Histol Histopathol. 1999; 14:1287–1294.
3. Ribatti D. Endogenous inhibitors of angiogenesis: a historical review. Leuk Res. 2009; 33:638–644.
4. Rezzola S, Belleri M, Gariano G, et al. In vitro and ex vivo retina angiogenesis assays. Angiogenesis. 2014; 17:429–442.
Article
5. Yoshida T, Gong J, Xu Z, et al. Inhibition of pathological retinal angiogenesis by the integrin αvβ3 antagonist tetraiodothyroacetic acid (tetrac). Exp Eye Res. 2012; 94:41–48.
Article
6. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003; 9:669–676.
Article
7. Siemerink MJ, Klaassen I, Van Noorden CJ, Schlingemann RO. Endothelial tip cells in ocular angiogenesis: potential target for anti-angiogenesis therapy. J Histochem Cytochem. 2013; 61:101–115.
8. Kim LA, D'Amore PA. A brief history of anti-VEGF for the treatment of ocular angiogenesis. Am J Pathol. 2012; 181:376–379.
Article
9. Hirschi KK, D'Amore PA. Pericytes in the microvasculature. Cardiovasc Res. 1996; 32:687–698.
Article
10. Hellstrom M, Gerhardt H, Kalen M, et al. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol. 2001; 153:543–553.
11. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 2003; 314:15–23.
Article
12. Stratman AN, Schwindt AE, Malotte KM, Davis GE. Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization. Blood. 2010; 116:4720–4730.
Article
13. Antonelli-Orlidge A, Saunders KB, Smith SR, D'Amore PA. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci U S A. 1989; 86:4544–4548.
Article
14. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997; 277:55–60.
Article
15. Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions. Circ Res. 2005; 97:512–523.
Article
16. Park SW, Yun JH, Kim JH, et al. Angiopoietin 2 induces pericyte apoptosis via α3β1 integrin signaling in diabetic retinopathy. Diabetes. 2014; 63:3057–3068.
Article
17. Connor KM, Krah NM, Dennison RJ, et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat Protoc. 2009; 4:1565–1573.
Article
18. Hewing NJ, Weskamp G, Vermaat J, et al. Intravitreal injection of TIMP3 or the EGFR inhibitor erlotinib offers protection from oxygen-induced retinopathy in mice. Invest Ophthalmol Vis Sci. 2013; 54:864–870.
Article
19. Kielczewski JL, Hu P, Shaw LC, et al. Novel protective properties of IGFBP-3 result in enhanced pericyte ensheathment, reduced microglial activation, increased microglial apoptosis, and neuronal protection after ischemic retinal injury. Am J Pathol. 2011; 178:1517–1528.
Article
20. Wilkinson-Berka JL, Deliyanti D, Rana I, et al. NADPH oxidase, NOX1, mediates vascular injury in ischemic retinopathy. Antioxid Redox Signal. 2014; 20:2726–2740.
Article
21. Zhao M, Shi X, Liang J, et al. Expression of pro- and anti-angiogenic isoforms of VEGF in the mouse model of oxygen-induced retinopathy. Exp Eye Res. 2011; 93:921–926.
Article
22. Smith LE, Wesolowski E, McLellan A, et al. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci. 1994; 35:101–111.
23. Mendel TA, Clabough EB, Kao DS, et al. Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy. PLoS One. 2013; 8:e65691.
Article
24. Park SW, Kim JH, Kim KE, et al. Beta-lapachone inhibits pathological retinal neovascularization in oxygen-induced retinopathy via regulation of HIF-1α. J Cell Mol Med. 2014; 18:875–884.
25. Kim J, Chung M, Kim S, et al. Engineering of a biomimetic pericyte-covered 3D microvascular network. PLoS One. 2015; 10:e0133880.
Article
26. Buch H, Vinding T, Nielsen NV. Prevalence and causes of visual impairment according to World Health Organization and United States criteria in an aged, urban Scandinavian population: the Copenhagen City Eye Study. Ophthalmology. 2001; 108:2347–2357.
Article
27. Wong WL, Su X, Li X, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health. 2014; 2:e106–e116.
Article
28. Feng Y, vom Hagen F, Pfister F, et al. Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression. Thromb Haemost. 2007; 97:99–108.
Article
29. Hughes S, Chan-Ling T. Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo. Invest Ophthalmol Vis Sci. 2004; 45:2795–2806.
Article
30. Sims DE. Diversity within pericytes. Clin Exp Pharmacol Physiol. 2000; 27:842–846.
Article
31. Frank RN, Turczyn TJ, Das A. Pericyte coverage of retinal and cerebral capillaries. Invest Ophthalmol Vis Sci. 1990; 31:999–1007.
32. Gerhardt H, Wolburg H, Redies C. N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken. Dev Dyn. 2000; 218:472–479.
Article
33. Patz A. Clinical and experimental studies on retinal neovascularization: XXXIX Edward Jackson memorial lecture. Am J Ophthalmol. 1982; 94:715–743.
34. Ishibashi T, Inomata H, Sakamoto T, Ryan SJ. Pericytes of newly formed vessels in experimental subretinal neovascularization. Arch Ophthalmol. 1995; 113:227–231.
Article
35. Salomon D, Ayalon O, Patel-King R, et al. Extrajunctional distribution of N-cadherin in cultured human endothelial cells. J Cell Sci. 1992; 102(Pt 1):7–17.
Article
36. Barbulovic-Nad I, Au SH, Wheeler AR. A microfluidic platform for complete mammalian cell culture. Lab Chip. 2010; 10:1536–1542.
Article
37. Kim S, Lee H, Chung M, Jeon NL. Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip. 2013; 13:1489–1500.
Article
38. Huh D, Hamilton GA, Ingber DE. From 3D cell culture to organs-on-chips. Trends Cell Biol. 2011; 21:745–754.
Article
Full Text Links
  • KJO
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2022 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr