Research ReportGalectin-3 enhances angiogenic and migratory potential of microglial cells via modulation of integrin linked kinase signaling
Highlights
► Galectin 3 has protective effects on microglia cells exposed to ischemic conditions. ► Galectin 3 promotes angiogenic and migratory potential under ischemic conditions. ► Galectin-3 mediates its effects through ILK signaling. ► Galectin-3 may be an important contributor for cerebral ischemic injury repair.
Introduction
Following cerebral ischemia, expression of neurotrophic factors, cytokines, and chemokines increase significantly. Many of these factors contribute to neuronal survival, migration, and angiogenesis that can promote self-repair (Jablonska and Lukomska, 2011, Kalluri and Dempsey, 2008, Lambertsen et al., 2012, Yan et al., 2009).
Angiogenesis and migration are tightly linked processes that are stimulated by specific tropic factors, chemokines, and cytokines (Bussolino et al., 1997, Comte et al., 2011, Hess and Allan, 2011, Ribatti et al., 2003, Thored et al., 2007, Vagima et al., 2011, Wang et al., 2011, Xiong et al., 2010). Particularly, the carbohydrate-binding proteins including galectin-3 (Gal-3) play a major role in cell growth, survival, angiogenesis, and motility (Boscher et al., 2012, Cay, 2012, Nangia-Makker et al., 2000, Markowska et al., 2010, Newlaczyl and Yu, 2011). Gal-3 is shown to induce endothelial cell morphogenesis, migration, and angiogenesis in various types of tumors (Nangia-Makker et al., 2000, Markowska et al., 2011, Yu, 2010). The Role of galectins in post ischemic angiogenesis and brain plasticity is not completely understood. Recent study reports that galectin-1 enhances the production of the cytokine BDNF and improves functional outcome in rats following cerebral ischemia (Qu et al., 2010). Increased Gal-3 was also observed after renal ischemia reperfusion in the rat (Vansthertem et al., 2010, Fernandes Bertocchi et al., 2008), liver ischemia and reperfusion (Lee and Song, 2002), and in microglia during neonatal hypoxic-ischemic brain injury (Doverhag et al., 2010). Early changes following ischemic injury involve activation of microglia that produce numerous growth factors and cyto/chemokines some known to be neuroprotective, and some neurotoxic (Davalos et al., 2005, Venneti et al., 2006, Weinstein et al., 2010, Sahota and Savitz, 2011). The main source of Gal-3 appears to be the microglia (Walther et al., 2000, Yan et al., 2009, Doverhag et al., 2010, Satoh et al., 2011, Lalancette-Hébert et al., 2012). We observed Gal-3 up-regulation in astrocytes as well, and showed that exogenous Gal-3 increases proliferation of endothelial and neural progenitor cells, and enhances microvessel density in ischemic rat brain (Yan et al., 2009).
Gal-3 acts through various signaling pathways including survival and integrin signaling pathways (Balan et al., 2012, Newlaczyl and Yu, 2011, Cay, 2012, Matarrese et al., 2000, Filer et al., 2009, Lei et al., 2009). Recent study has shown that Gal-3 modulates bFGF and VEGF mediated αv β 3 integrin signaling (Markowska et al., 2010). Integrin linked kinase (ILK), a serine/threonine kinase is known to promote endothelial cell migration, proliferation and tube formation in vitro, and up-regulates VEGF levels in human scar fibroblasts and gastric cancer (McDonald et al., 2008, Mi et al., 2011). The effects ILK is mediated by its downstream signaling pathways, including mitogen activated protein kinase (MAPK-ERK1/2), and phosphoinositol-3 kinase (PI3K-Akt) (Eke et al., 2009, Krasilnikov, 2000, Legate et al., 2006, Wani et al., 2011, Yoganathan et al., 2000). The role of Gal-3 and the mechanism with which it mediates its function under ischemic conditions is not well understood.
In this study, we show that Gal-3 increases the viability of microglia BV2 cells subjected to OGD/re-oxygenation through activation of phos-Akt. Furthermore, Gal-3 promoted the formation of pro-angiogenic structures and in vitro migratory potential of BV2 microglia. These actions of Gal-3 were mediated through integrin-linked kinase (ILK) signaling as shown by impaired angiogenesis and migration of BV2 cells following siRNA mediated silencing of ILK expression. Taken together, our studies support a role for Gal-3 in promoting angiogenesis and microglial cell migration, the critical processes that potentially contribute to post stroke repair.
Section snippets
Gal-3 increases the survival of BV2 microglial cells exposed to OGD
Ischemic micro-environment can lead to decreased cell viability. Although controversial, the beneficial effects of microglia have been recently recognized. We examined the protective effects of Gal-3 on BV2 microglia under in vitro ischemia/re-oxygenation injury. The viability of BV2 microglia was significantly increased by Gal-3 treatment in a dose dependent manner. Gal-3 (5 μg/ml) resulted in increased number of viable cells subjected to OGD, by about 30–35% as compared to untreated cells (
Discussion
Ischemic injury changes microenvironment by altering inflammatory cytokines. Understanding the functional consequences and molecular mechanisms of these cytokines may provide clues for improving therapeutic strategies for post stroke repair. Post ischemic repair requires concerted actions of survival, neo-angiogenesis, and recruitment of progenitor cells and microglia to injured site. Although the cytokine Gal-3 is well recognized to modulate multiple physiological functions, its role in
Cell culture
Murine BV-2 microglia cell line developed by Dr. V. Bocchini was a generous gift from Dr. Grace Y Sun (University of Missouri, Columbia, MO). Human Umbilical Vein Endothelial Cells (HUVEC) was purchased from American Type Culture Collection (ATCC; Manassas, VA). The BV2 cells were grown in DMEM medium containing 10% fetal bovine serum, 0.1 mM non-essential amino acids, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. HUVEC cells were grown in a complete medium with growth supplements (Cell
Disclosures
The authors have no conflict of interest and nothing to disclose.
Acknowledgments
This work was supported by NIH-5R01NS063959 (PI Dempsey) and by funding from the Department of Neurosurgery, University of Wisconsin, Madison WI.
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