Abstract
Purpose:
Pentraxin 3 (PTX3), an inducible secreted protein in most cell types, is crucial for innate immunity, tissue inflammation, and stability of heavy chain-hyaluronan (HC-HA) complex in cumulus oophorus complex. We have reported that HC-HA purified from human amniotic membrane (AM) is responsible for AM’s therapeutic actions and constitutively produced by AM cells. Furthermore, upregulation of PTX3 to suppress expression and activation of matrix metalloproteinases 1 and 3 is involved in conjunctivochalasis fibroblasts. Herein, we further investigated whether PTX3 is also in HC-HA complex produced by AM, which is used as a surgical graft for treating conjunctivochalasis.
Methods:
Expression of PTX3 was determined by immunostaining of AM tissue, and by RT-PCR and Western blot of supernatants and lysates of primary AM epithelial (AMEC) and stromal cells (AMSC) treated with or without TNF, IL-1β, or PTX3 siRNA in a low-serum medium. HC-HA complexes were isolated from AM PBS and guanidine HCl extracts by sequential CsCl ultracentrifugation in 6 M guanidince HCl, and further characterized by Western blot with or without hyaluronidase (HAase) digestion.
Results:
Strong positive PTX3 staining was found in the compact stromal layer and the apical surface of the epithelium in the AM. Like conjunctivochalasis and skin fibroblasts, PTX3 mRNA was expressed by resting AMEC and AMSC and further upregulated by TNF or IL-1β. Unlike strict contingence upon IL-1β in cultured human skin and conjunctivochalasis fibroblasts, expression of PTX3 protein was constitutive by resting AMEC and AMSC, while its secretion was reduced by TNF and completely inhibited by IL-1β. Secretion of PTX3 protein under stimulation of TNF was downregulated by siRNA in conjunctivochalasis fibroblasts but not in AMEC and AMSC. PTX3 was released from HC-HA complex by HAase in both water-soluble and water-insoluble HC-HA complex purified from AM.
Conclusions:
PTX3 is an integral component of HC-HA complex purified from both water-soluble and water-insoluble AM extracts. Expression and secretion of PTX3 depends on pro-inflammatory cytokines in skin fibroblasts, but is constitutive in resting AM epithelial and stromal cells. Thus, HC-HA complex that contains PTX3 is uniquely present in AM and is responsible for AM’s clinical applications.
Keywords: 480 cornea: basic science •
519 extracellular matrix •
474 conjunctiva