Astrocyte-Derived Sonic Hedgehog Contributes to Angiogenesis in Brain Microvascular Endothelial Cells via RhoA/ROCK Pathway After Oxygen–Glucose Deprivation

Abstract

The human adult brain possesses intriguing plasticity, including neurogenesis and angiogenesis, which may be mediated by the activated sonic hedgehog (Shh). By employing a coculture system, brain microvascular endothelial cells (BMECs) cocultured with astrocytes, which were incubated under oxygen-glucose deprivation (OGD) condition, we tested the hypothesis that Shh secreted by OGD-activated astrocytes promotes cerebral angiogenesis following ischemia. The results of this study demonstrated that Shh was mainly secreted by astrocytes and the secretion was significantly upregulated after OGD. The proliferation, migration, and tube formation of BMECs cocultured with astrocytes after OGD were significantly enhanced, but cyclopamine (a Shh antagonist) or 5E1 (an antibody of Shh) reversed the change. Furthermore, silencing Ras homolog gene family, member A (RhoA) of BMECs by RNAi and blocking Rho-dependent kinase (ROCK) by Y27632, a specific antagonist of ROCK, suppressed the upregulation of proliferation, migration, and tube formation of BMECs after OGD. These findings suggested that Shh derived from activated astrocytes stimulated RhoA/ROCK pathway in BMECs after OGD, which might be involved in angiogenesis in vitro.

Astrocytes, therapeutic targets for neuroprotection and neurorestoration in ischemic stroke

“…Down-regulation of Shh expression in astrocytes disrupts the BBB (Alvarez et al , 2011), suggesting that stimulation of astrocytic Shh production could promote restoration of BBB integrity. Shh further induces upregulation of angiopoietin-1 in astrocytes, which is necessary for vessel maturation (He et al , 2013). …”

The Role of Smoothened and Hh Signaling in Neovascularization

“…Altogether these data suggest a dual signaling of Hh proteins; whereas Hh proteins prime the initial steps of angiogenesis, mostly cytoskeletal activation, cell elongation, and extended survival by acting through noncanonical signals, the indirect effects of Hh on Gli-dependent secretion of VEGF and Ang1 and 2 by mesenchymal cells may provide the additional contribution required to enhance endothelial cell proliferation, migration, survival, and vessel maturation [127]. Similar mechanism implicating RhoA-/Rho-associated protein kinase (ROCK) pathway has been described in a co-culture model of brain microvascular endothelial cells and astrocytes during oxygen-glucose deprivation [128]. In fact, after oxygen-glucose deprivation, astrocytes released Shh which directly promoted angiogenesis via the RhoA/ROCK pathway.…”

The Hedgehog Signaling Pathway in the Ischemic Heart, Brain, and Skeletal Muscle

“…Middle cerebral artery occlusion is associated with increased mRNA encoding both Shh and its transcription factor Gli1, already a few hours after induction of brain ischemia [38]. By employing a co-culture system of brain microvascular endothelial cells (BMECs) and astrocytes, incubated under oxygen-glucose deprivation (OGD) condition, it has been demonstrated that Shh is secreted by activated astrocytes and promotes proliferation, migration, and tube formation of BMECs [39]. In this assay, proliferation, migration, and tube formation of BMECs may be suppressed by silencing the Ras homolog gene family member A (RhoA) and blocking Rho-dependent kinase (ROCK) [39].…”

“…By employing a co-culture system of brain microvascular endothelial cells (BMECs) and astrocytes, incubated under oxygen-glucose deprivation (OGD) condition, it has been demonstrated that Shh is secreted by activated astrocytes and promotes proliferation, migration, and tube formation of BMECs [39]. In this assay, proliferation, migration, and tube formation of BMECs may be suppressed by silencing the Ras homolog gene family member A (RhoA) and blocking Rho-dependent kinase (ROCK) [39]. Not only ODG, but also oxidative stress has been shown to have the ability of activating the Shh pathway in cultured cortical neurons [40].…”