Neurotensin, substance P, and insulin enhance cell migration

Abstract

Neurotensin, substance P, and insulin have been demonstrated to improve wound healing in vivo. However, the mechanism behind their effect is still not fully understood. This study investigates the effects leading to enhanced scratch closure by these peptides in vitro. The skin keratinocyte cell line, HaCaT, was used to test scratch closure effects of the peptides and alterations of cytokine levels. HUVEC cells were used to test the angiogenic effect of the peptides. Furthermore, clinical isolates of Staphylococcus lugdunensis were used to examine the potential antimicrobial activity of each peptide. Our results demonstrate that neurotensin, substance P, and insulin had significant migratory effects in scratch assays were neurotensin had the lowest effect. Furthermore, we investigated use of the peptides in combination. When substance P was used in combination with neurotensin, the cell migratory capacity was decreased, and the peptides showed a negative correlation (r = -0.298, P < .001). Neurotensin and insulin significantly increased levels of monocyte chemoattractant protein-1 (P < .001) secreted from white blood cells, whereas substance P showed a tendency. Interestingly, neurotensin increased the level of monocyte chemoattractant protein-1 significantly compared to substance P (P < .01). Additionally, the peptides decreased TNFα mRNA levels (P < .001) in HaCaT cells, whereas only neurotensin and insulin decreased IL-8 mRNA (P < .001) but had no significant effect on IL-6 mRNA levels. Surprisingly, substance P increased IL-6 mRNA 9-fold (P < .001). Furthermore, we demonstrate that the peptides increased angiogenesis in the HUVEC cells (P < .001). Finally, S. lugdunensis isolates were not susceptible to the peptides. We demonstrate that the peptides worked differently on HaCaT cells, but substance P acted differently than neurotensin on cytokine levels expression as well as on migration of HaCaT cells. On the contrary, neurotensin and insulin worked similarly. All of these aspects are crucial for proper wound healing, and the results suggest multiple mechanisms for wound-healing properties of these peptides.

Halogenation as a tool to tune antimicrobial activity of peptoids

“…An immortalized human keratinocytes (HaCaT) cell line (Gift from David G. Naym at Bispebjerg Hospital) were cultured to ∼90% confluence after 21–25 h of growth at 5% CO 2 and 37 °C as previously 48 . In brief, the cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 μg/mL), purchased from Sigma-Aldrich (St. Louis, MO, United States).…”

Immunomodulatory potential of Nisin A with application in wound healing

“…The wells were washed with 250 μl PBS, and a scratch was manually performed with a 200 μl pipet tip, during the second wash. After removing the PBS, Nisin A was added in a concentration of 25 μg/mL in media. The plate was incubated at 37 °C and in 5% CO 2 for 24 hours in the automated optical camera system oCelloScope (BioSense, Denmark), where migration of the cells was captured every hour and processed by the UniExplorer software (version 8.0.0.7144 (RL3)), as previously demonstrated by our group . For the HUVEC cells, the precoating was done previously to seeding the cells.…”

“…The plate was incubated at 37 C and in 5% CO 2 for 24 hours in the automated optical camera system oCelloScope (BioSense, Denmark), where migration of the cells was captured every hour and processed by the UniExplorer software (version 8.0.0.7144 (RL3)), as previously demonstrated by our group. 17,18 For the HUVEC cells, the precoating was done previously to seeding the cells. Cells treated with free amino acids in the same mole-ratio as in Nisin A were used as a negative control and epithelial growth factor (EGF) was used as a positive control at 500 ng/ml.…”

Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides

“…The 96-well plates were supplied by Corning Costar (Sigma-Aldrich, Brøndby, Denmark). Cell viability assessment was performed on cell monolayers grown to ∼90% confluence in 96-well plates by using the MTS/PMS assay as previously described 85 . Briefly, the adhered cells were washed with PBS solution (37 °C) (ThermoFisher Scientific, Roskilde) and exposed for 1 h at 37 °C to 100 μL of peptides dissolved in the medium also used for culturing of the cell line (at concentrations in the range 0-1,000 μg/mL).…”