ABSTRACT
Small interfering RNA (siRNA) was developed as a novel tool to inhibit gene function in human disease. The aim of the present study was to modify the function of NF-kappaB in airway epithelial cells by application of siRNA. 1HAEo cells were transfected with siRNA directed to the p65 subunit of NF- kappaB (siRNA.p65). Application of siRNA.p65 caused decreased levels of p65 mRNA or protein after 72 hours, as determined by quantitative RT-PCR or Western blot analysis. The tumor necrosis factor- alpha (TNF-alpha)-induced release of interleukin-6 (IL-6) and IL-8 was significantly inhibited by the application of siRNA.p65. Well-differentiated primary cells were resistant to transfection with siRNA.p65. However, when undifferentiated primary cells were transfected, an effect of the siRNA could still be observed when the cells were differentiated in an air-liquid interface culture system. In conclusion, siRNA can be used to regulate the activity of NF-kappaB in airway epithelial cells.
Subject(s)
Bronchi/metabolism , Gene Expression Regulation , RNA Interference , RNA, Small Interfering/pharmacology , Respiratory Mucosa/metabolism , Bronchi/drug effects , Gene Expression Regulation/drug effects , Humans , Inflammation Mediators/physiology , NF-kappa B/biosynthesis , NF-kappa B/genetics , Respiratory Mucosa/drug effectsABSTRACT
Antimicrobial peptides are endogenous antibiotics that directly inactivate microorganisms and in addition have a variety of receptor-mediated functions. LL-37/hCAP-18 is the only cathelicidin found in humans and is involved in angiogenesis and regulation of the innate immune system. The aim of the present study was to characterize the role of the peptide LL-37 in the regulation of wound closure of the airway epithelium in the cell line NCI-H292 and primary airway epithelial cells. LL-37 stimulated healing of mechanically induced wounds in monolayers of the cell line and in differentiated primary airway epithelium. This effect was detectable at concentrations of 5 mug/ml in NCI-H292 and 1 mug/ml in primary cells. The effect of LL-37 on wound healing was dependent on the presence of serum. LL-37 induced cell proliferation and migration of NCI-H292 cells. Inhibitor studies in the wound closure and proliferation assays indicated that the effects caused by LL-37 are mediated through epidermal growth factor receptor, a G protein-coupled receptor, and MAP/extracellular regulated kinase. In conclusion, LL-37 induces wound healing, proliferation, and migration of airway epithelial cells. The peptide is likely involved in the regulation of tissue homeostasis in the airways.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Bronchi/drug effects , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/physiology , Antimicrobial Cationic Peptides/toxicity , Base Sequence , Bronchi/cytology , Bronchi/injuries , Bronchi/metabolism , Cell Line , Cell Proliferation/drug effects , DNA, Complementary/genetics , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression/drug effects , Humans , Receptors, Formyl Peptide/genetics , Receptors, Leukotriene B4 , Receptors, Lipoxin/genetics , Receptors, Purinergic P2/genetics , Signal Transduction/drug effects , Wound Healing/drug effects , Wound Healing/genetics , Wound Healing/physiology , CathelicidinsABSTRACT
Rat amylin differs from human amylin (hIAPP) in that it lacks a fibril-forming capacity. As a consequence, toxic effects have been reported for human but not for rat amylin. This report demonstrates how a mercury contamination of commercial rat amylin imitates peptide-related vasoactive and cytotoxic effects on preparations of isolated blood vessels. The source of mercury contamination was believed related to the peptide synthesis. Thiol groups of cysteine-containing peptides are often protected by acetamidomethyl (Acm) which is cleaved by mercuric acetate.
Subject(s)
Amyloid/pharmacology , Anti-Ulcer Agents/pharmacology , Mercury/metabolism , Pulmonary Artery/drug effects , Acetylcholine/metabolism , Amyloid/metabolism , Amyloid/toxicity , Animals , Anti-Ulcer Agents/metabolism , Anti-Ulcer Agents/toxicity , Californium/metabolism , Humans , Islet Amyloid Polypeptide , Male , Mass Spectrometry , Mercury/toxicity , RatsABSTRACT
Mercury and its derivatives are known to constrict vascular smooth muscle cells. However, little is known about the role of endothelial cells in mercury-induced vasoreactivity. Using isolated, norepinephrine preconstricted rat aorta and pulmonary artery rings with intact endothelium, we demonstrate that mercury chloride (HgCl2) induces an endothelial-dependent vasorelaxation which was totally blocked by the nitric oxide inhibitor L-NAME. Besides this vasorelaxant effect, treatment with HgCl2 resulted in functional and morphological alterations of the endothelial cells. On aortic rings, endothelial cells were partly lifted from the basal membrane when incubated for 20 min in HgCl2 (10(-7) M)-containing buffer. At a concentration of 10(-6) M, the endothelial cells were completely denuded and acetylcholine vasorelaxation was abolished. Endothelial cell structure and function was preserved by incubating the vessels in HgCl2-containing rat blood instead of buffer. We conclude that HgCl2 induces an endothelial-dependent vasorelaxation and alters structure and function of vascular endothelial cells.
