Detection of Pro- and Antiangiogenic Factors in the Human Sclera

ABSTRACT

Purpose: Avascular tissues can be used to identify antilymph- or antihemangiogenic factors. The human sclera—the outer covering layer of the eye, lacks lymphatic vessels and contains only a superficial network of blood vessels and was used here to identify endogenous antiangiogenic factors.

Methods: Expression levels of a panel of 96 known pro- and antiangiogenic factors were analyzed in 12 scleral or conjunctival control samples from normal human donors using real-time PCR. In vitro, scleral homogenate was cocultured with blood- and lymphatic endothelial cells (BECs and LECs) and immunohistochemistry was performed of scleral fibroblasts and BECs.

Results: Three antiangiogenic factors were significantly upregulated in the human sclera compared to the conjunctiva, including FBLN5 (fibulin 5), SERPINF1 (serpin peptidase inhibitor, clade F, member 1 = pigment epithelium derived factor) and TIMP2 (Tissue inhibitor of metalloproteinases 2). Six proangiogenic factors were significantly downregulated in the sclera, including FLT4 (Fms-related tyrosine kinase 4=VEGF-R3), HGF (hepatocyte growth factor), KIT (CD117 / c-kit), PROX1 (prospero homeobox 1), SEMA3F (semaphorin-3F) and TGFA (transforming growth factor alpha). In vitro, scleral homogenate inhibited the growth of both BECs and LECs. Immunohistochemistry labeling of three major antiangiogenic factors from scleral tissue confirmed TIMP3 and PEDF expression both in scleral fibroblasts and in blood endothelial cells, whereas TIMP2 was not detectable.

Conclusion: Balancing anti- and proangiogenic factors actively regulates human scleral avascularity, inhibits endothelial cell growth in vitro, and thus may help maintaining the vascular privilege of the inner eye.

KEYWORDS:

• Sclera
• connective tissue
• (Lymph)-angiogenesis
• conjunctiva
• real-time PCR
• PEDF
• TIMP

Acknowledgments

The authors thank Sabine Hackbarth, Monika Lindner, and Günther Simons from the Eyebank of the University of Cologne for their support.

Compliance with Ethical Standards

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Ethic approval: All procedures performed involving human tissues were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable standards.

Informed consent: Informed consent was obtained from all individual participants or their family members included in the study.

Disclosure statement

The authors reported no potential conflict of interest.

Additional information

Funding

This work was supported by DFG (Deutsche Forschungs Gemeinschaft) ”Lymph Angiogenesis and Cellular Immunity In Inflammatory Diseases of the Eye” [grant number FOR 2240 to CC, DH, and LMH]; [grant number HO 5556/1-1 to DH], [grant number HE 6743/2-1 to LMH], [grant number HE 6743/3-1 to LMH]; [grant number CU 47/6-1 to CC], [grant number CU47/9-1 to CC], [grant number CU47/12-1 to CC]; German Cancer Aid (to LMH and CC), GEROK Program University of Cologne (to SLS, DH, and LMH); EU COST BM1302 “Joining Forces in Corneal Regeneration” (to CC and DH).