Abstract
Degradation products of elastin, i.e. elastin-derived peptides (EDPs), are involved in various physiological and pathological processes. EDPs are detectable in cerebrospinal fluid in healthy people and in patients after ischemic stroke. However, to date, no studies concerning the role of EDP in the nervous system were conducted. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play important roles during the repair phases of cerebral ischemia, particularly during angiogenesis and reestablishment of cerebral blood flow. Therefore, the aim of this study was to investigate the impact of the specific elastin-derived peptide VGVAPG on Mmp-2, -9 and Timp-1, -2, -3 and -4 mRNA expression in mouse cortical glial cells in vitro. Primary glial cells were maintained in DMEM/F12 without phenol red supplemented with 10% fetal bovine serum and the cells were exposed to 50 nM, 1 and 50 μM of the VGVAPG peptide. After 3 and 6 h of exposition to the peptide, expression of Mmp-2, -9 and Timp-1, -2, -3 and -4 mRNA was measured. Moreover, siRNA gene knockdown, cytotoxicity and apoptosis measurement were included in our experiments, which showed that VGVAPG in a wide range of concentrations exhibited neither proapoptotic nor cytotoxic properties in mouse glial cells in vitro. The peptides enhanced mRNA expression of Timp-2 and Timp-3 genes in an elastin-binding protein (EBP)-dependent manner. However, changes in mRNA expression of Mmp-2, Mmp-9 and Timp-4 were partially EBP-dependent. The decrease in mRNA expression of Timp-1 was EBP-independent. However, further studies underlying the VGVAPG peptide’s mechanism of action in the nervous system are necessary.
Highlights
Elastin is an essential protein in mammalian organisms and provides elasticity to many connective tissues such as the major arteries, lung, cartilage, elastic ligaments and skin
After 24 and 48 h of exposure of primary mouse glial cells to the studied VGVAPG peptide in concentrations ranging from 100 pM to 100 μM, we observed a lack of stimulation of lactate dehydrogenase (LDH) release (Fig. 1a)
After 24 and 48 h of exposure of primary mouse glial cells to the studied VGVAPG peptide in concentrations ranging from 100 pM to 100 μM, we observed a lack of stimulation of caspase-3 activity (Fig. 1b)
Summary
Elastin is an essential protein in mammalian organisms and provides elasticity to many connective tissues such as the major arteries, lung, cartilage, elastic ligaments and skin. Products of proteolytic degradation of elastin, namely elastin-derived peptides (EDPs), are involved in various physiological and pathological processes (Gmiński et al 1992, 1993). In different physiological and pathophysiological conditions, such as inflammation or atherosclerosis, elastin is prone to proteolytic degradation which frees Val-Gly-Val-Ala-ProGly (VGVAPG)-containing fragments (Lombard et al 2006; O’Rourke 2007). The VGVAPG (498.58 molecular weight (MW)) hexapeptide is repeated multiple times in elastin molecules and binds to 67-kDa elastin-binding protein (EBP) with high affinity (Blood et al 1988; Senior et al 1984). The VGVAPG peptide exhibits strong chemotactic properties in the murine lung carcinoma cell line (M27) and facilitates the invasion of human melanoma cells (WM35 and HT168-M1) (Blood et al 1988; Pocza et al 2008). In addition to its chemotactic properties, it has been shown that EDPs or the VGVAPG peptide upregulated the expression of different metalloproteinases (Floquet et al 2004; Siemianowicz et al 2010)
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