Abstract
ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces α2β1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.
Highlights
Function and maintenance of tissue integrity depend on specific interactions of cells with the surrounding extracellular matrix (ECM)
It was previously demonstrated that ALT-C, an α2β1-integrin ligand, induces vascular endothelial growth factor (VEGF) expression in fibroblasts and endothelial cell viability in doses as low as 10 nM [19]
In the presence of collagen I, increased protein concentrations were require to provoke similar results (150 nM). This effect was influenced by incubation time and was more consistent after 48 hours. These results suggest that α2β1 integrin may be relevant for muscle cell viability
Summary
Function and maintenance of tissue integrity depend on specific interactions of cells with the surrounding extracellular matrix (ECM). Disintegrins were first described in 1989 as a protein group with low molecular weight (5 to 9 kDa), that interact with integrin receptors on the cell surface [14,15,16] These peptides represent a family of cysteine-rich proteins, isolated from snake venoms, and are known to inhibit cell-to-matrix and cell-to-cell interactions mediated by integrins [2, 17]. These proteins are larger than RGD disintegrins (about 30 kDa) and have an extra C-terminal, cysteine-rich domain They do not bind to αIIbβ, α5β1 or αvβ integrins, but interact with the collagen receptor, α2β1 integrin, inhibiting cell adhesion to collagen I. The use of integrin-binding molecules that up-regulate the expression of growth factors may be useful in these studies This is the first demonstration of the influence of a disintegrin on myoblast proliferation and myosin gene expression
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