Heparin modulates the growth and adherence and augments the growth‐inhibitory action of TNF‐α on cultured human keratinocytes

Abstract

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.

Heparan Sulfate Plays a Central Role in a Dynamic in Vitro Model of Protein-losing Enteropathy

“…HS binds to both IFN␥ (25,33) and TNF␣ (34,35). In most cases, cytokines as well as growth factors employ cell-associated HS to enhance their signal, as previously shown for TNF␣ (34). In contrast, we report that binding to HS seems to lower the concentration of available TNF␣ as well as of IFN␥, whereas loss of cell-associated HS enhances their activities.…”

“…Heparin binds to both IFN␥ (25,33) and TNF␣ (34,35) and inactivates them (25,34), which alleviates protein leakage in our model (Fig. 6A).…”

“…3A). HS binds to both IFN␥ (25,33) and TNF␣ (34,35). In most cases, cytokines as well as growth factors employ cell-associated HS to enhance their signal, as previously shown for TNF␣ (34).…”

Heparin Cryoprecipitation Reduces Plasma Levels of Non-Traditional Risk Factors for Atherosclerosis in vitro

“…TNF-␣ induces thrombotic and inflammatory reactions in endothelial cells, and also induces production of IL-6 by these cells [34] . We suggest that the highly significant reduction in TNF-␣ from HD plasma may result from its binding to heparin [35] , forming a complex that is then precipitated by the HCP method.…”

Heparin Promotes Suspension Adaptation Process of CHO–TS28 Cells by Eliminating Cell Aggregation

“…In contrast, heparin has been successfully applied in suspension cultures of TN 5B-1-4 insect cells [14] and HEK293 cell [15,16] in virus-cell culture systems that have culture phases short in duration and on a small scale. Herein, heparin plays important roles of anti-aggregation by inhibiting cell-to-cell adhesion [17,18]. Until recently, there was limited data about its application in CHO cell suspension adaptation.…”