Effects of Exogenous Extracellular Matrix Proteins on the Reattachment of Retinal Pigment Epithelial Cells

Kwang Soo Kim; Yu Cheol Kim; Se Jin Jeon

doi:10.3341/jkos.2007.48.11.1537

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Kim, Kim, and Jeon: Effects of Exogenous Extracellular Matrix Proteins on the Reattachment of Retinal Pigment Epithelial Cells

Original Article

J Korean Ophthalmol Soc 2007;48(11):1537-1547.

Published online: 28 August 2007

DOI: https://doi.org/10.3341/jkos.2007.48.11.1537

Effects of Exogenous Extracellular Matrix Proteins on the Reattachment of Retinal Pigment Epithelial Cells

Kwang Soo Kim, M.D.^1,, Yu Cheol Kim, M.D.¹, Se Jin Jeon, M.D.²

¹Department of Ophthalmology, Keimyung University, School of Medicine, Daegu, Korea

²Saebit Eye Clinic, Daegu, Korea

Address reprint requests to Kwang Soo Kim, M.D., Department of Ophthalmology, Dongsan Medical Center, Keimyung University, #194 Dongsan-dong, Jung-gu, Daegu 700-712, Korea, Tel: 82-53-250-7706, Fax: 82-53-250-7705, E-mail: kimks@dsmc.or.kr

Received 2 February 2006 Accepted 24 July 2007

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To evaluate the effect of adding exogenous extracellular matrix (ECM) proteins on the reattachment of retinal pigment epithelium (RPE) to the damaged surface of Bruch's membrane (BM).

Methods

Porcine BM explants were divided into six groups: BMs with an intact basal lamina (bl-BM) and five damaged BMs (d-BM: bare & four ECM-coated). The d-BM was coated with ECM proteins (either fibronectin, laminin, collagen IV, or all). Primary RPE sheets were plated and cultured for each group of BM explants. The attached live cells were counted and examined with a scanning electron microscope after three days, as well as at 1, 2 and 4 weeks.

Results

The RPE reattachment rate was highest in bl-BM and lowest in uncoated d-BM. ECM-coated groups showed a lower reattachment rate than bl-BM, but when compared with the uncoated group, the reattachment rate was significantly increased (p<0.05). ECM-exposure time did not influence the reattachment rate of any of the groups. RPE cells plated on bl-BMs and ECM-coated d-BMs attached and proliferated well and achieved confluence over time. Even though most cells were flat and large in shape, some cells revealed a good morphology with microvilli on their surface. On the other hand, only some of the RPE sheets plated on the uncoated d-BM attached loosely and most cells remained round and clumped.

Conclusions

These results show that the addition of ECM proteins may increase the ability of RPE cells to reattach to the damaged BM surface, which would likely create a good morphology.

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Keywords: Bruch's membrane, Exogenous extracellular matrix proteins, Retinal pigment epithelial cells

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Figure 1.

Reattachment rate of the retinal pigment epithelial (RPE) cells onto the Bruch's membrane (BM).

BL-BM=BM with basal lamina; B-BM=bare (ECM-uncoated); FN-BM=FN-coated; LN-BM=laminin-coated; CL-BM=collagen IV-coated; C-BM=ECM cocktail-coated.

Figure 2.

Scanning electron microscopic findings of RPE sheets cultured on BMs 3 days after plating.

(A) BM with BL (×250), (B) bare d-BM (×300), (C) FN-coated BM (×90), (D) LN-coated BM (×180), (E) CL-coated BM (×250), (F) ECM cocktail-coated BM (×180). RPE cells were readily reattached & flattened except on uncoated bare BM. BM =Bruch's membrane; BL=basal lamina; d-BM=damaged BM; FN=fibronectin; LN=laminin; CL=collagen IV; ECM=extracellular matrix.

Figure 3.

Scanning electron microscopic findings of RPE sheets cultured on BMs 1 week after plating.

(A) BM with BL (×90), (B) flat peripheral cells in A (×400), (C) bare d-BM (×500), (D) FN-coated BM (×180), (E) LN-coated BM (×180), (F) CL-coated BM (×130), RPE cells were much flattened and spreaded out on BM with BL & ECM coating. RPE cells on uncoated bare BM showed still round in shape. RPE=retinal pigment epithelium; BM=Bruch's membrane; BL=basal lamina; d-BM=damaged BM; FN=fibronectin; LN=laminin; CL=collagen IV; ECM=extracellular matrix.

Figure 4.

Scanning electron microscopic findings of RPE sheets cultured on BMs 2 weeks after plating.

(A & B) BM with BL (×30, ×90), (C & D) bare d-BM (×30, ×250), (E & F) FN-coated BM (×30, ×200), (G & H) LN-coated BM (×30, ×70). RPE cells were getting to confluence except on uncoated BM. Note round cells on bare d-BM (D) & fibroblast-like cells on ECM-coated BM(F, H). RPE = retinal pigment epithelium; BM = Bruch's membrane; BL = basal lamina; d-BM = damaged BM; FN = fibronectin; LN = laminin; CL = collagen IV; ECM = extracellular matrix.

Figure 5.

Scanning electron microscopic findings of RPE sheets cultured on BMs 4 weeks after plating.

(A & B) BM with BL (×200, ×500), (C & D) uncoated bare d-BM (×70, ×300), (E & F) FN-coated BM (×500), (G & H) LN-coated BM (×500), (I & J) CL-coated BM (×500, ×200). Note the central dome-shaped polygonal cells (A, E, G, I) and peripheral flat cells (B, F, H). Fibroblast-like cells and round cells were intermingled (J). On bare d-BM, RPE cells did not get confluence and most cells transformed to fibroblast-like cells. RPE = retinal pigment epithelium; BM = Bruch's membrane; BL = basal lamina; d-BM = damaged BM; FN = fibronectin; LN = laminin; CL = collagen IV.

Table 1.

Reattachment rate of the retinal pigment epithelium on the Bruch's membrane

ECM^* time(min)	Type of treated Bruch's membrane and its reattachment rate(%)
ECM^* time(min)	BL^†	Bare^§	Fibrinogen	Laminin	CL-IV^‡	Cocktail
30	39.9±8.5	19.5±8.2	30.3± 13.3	32.0±11.2	27.7±6.6	28.1±7.9
60	52.0± 12.9	18.0±9.9	30.2± 15.5	28.1±14.0	24.7±3.9	27.5±6.6
120	50.5±13.2	16.0±10.8	34.1±11.7	29.1±9.9	37.1±11.5	30.0±4.3

^* ECM = extracellular matrix.

^† BL = basal lamina.

^‡ CL-IV = collagen-IV.

^§ p<0.05 by Student's t-test.

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