Abstract
AimsSphingosine kinase 1 (Sphk1) and the signaling molecule sphingosine‐1‐phosphate (S1P) are known to be key regulators of a variety of important biological processes, such as neovascularization. Nitric oxide (NO) is also known to play a role in vasoactive properties, whether Sphk1/S1P signaling is able to alter angiogenesis in the context of cerebral ischemia‐reperfusion injury (IRI), and whether such activity is linked with NO production, however, remains uncertain.MethodsWe used immunofluorescence to detect the expression of Sphk1 and NOS in cerebral epithelial cells (EC) after IR or oxygen‐glucose deprivation (OGDR). Western blotting was used to detect the Sphk1 and NOS protein levels in brain tissues or HBMECs. Adenovirus transfection was used to inhibit Sphk1 and NOS. An NO kit was used to detect NO contents in brain tissues and epithelial cells. Tube formation assays were conducted to measure angiogenesis.ResultsWe determined that EC used in a model of cerebral IRI expressed Sphk1, and that inhibiting this expression led to decreased expression of two isoforms of NO synthase (eNOS and iNOS), as well as to decrease neovascularization density and NO production following injury. In HBMECs, knocking down Sphk1 markedly reduced NO production owing to reduced eNOS activity, and inhibiting eNOS directly similarly decreased NO production in a manner which could be reversed via exogenously treating cells with S1P. We further found that knocking down Sphk1 reduced HBMEC eNOS expression, in addition to decreasing the adhesion, migration, and tube formation abilities of these cells under OGDR conditions.ConclusionsBased on these results, we therefore postulate that Sphk1/S1P signaling is able to mediate angiogenesis following cerebral IRI via the regulation of eNOS activity and NO production. As such, targeting these pathways may potentially represent a novel means of improving patient prognosis in those suffering from cerebral IRI.
Highlights
Stroke remains the most common cause of death and disability worldwide,[1] with cerebral ischemia-reperfusion injuries (IRIs) having the potential to induce additional debilitating secondary damage
This indicates that Sphingosine kinase 1 (Sphk1)/S1P signaling plays a key role in regulating eNOS induction and angiogenesis in this in vitro oxygen-glucose deprivation (OGDR) model
Sphk1-mediated S1P production is known to be an essential mediator of microglial proinflammatory cytokine production.[15]
Summary
Stroke remains the most common cause of death and disability worldwide,[1] with cerebral ischemia-reperfusion injuries (IRIs) having the potential to induce additional debilitating secondary damage. Nitric oxide (NO) is a key vasoactive signaling molecule which can modulate local blood flow via promoting vasodilation and reducing cerebral vascular resistance.[8,9] The NO signaling molecule is a highly reactive and short-lived small molecule that can be produced by three forms of NO synthase (NOS) enzymes in specific contexts, with ECs and perivascular nitrergic neurons being primary sources of NO production.[10] When release in the brain, NO can be both beneficial and harmful depending on the specific context, with factors including the NOS isoform induced, the cellular source of NO, and the time relative to IRI being critical for determining its relative harm or benefit. The S1P receptor, which is induced in cerebral microvessels early after I/R injury, potently regulates brain endothelium responses to ischemic and inflammatory injury.[24] In addition, previous work in HUVEC cells has shown that knocking down Sphk[1] can markedly impair their proliferation and migration.[25] How endothelial Sphk1/S1P signaling impacts stroke prognosis via neovascularization and collateral establishment, remain uncertain. NO synthesis has not been adequately studied in the context of the role of Sphk1/ S1P on cerebral ischemia prognosis, and our research has the potential to identify novel therapeutic avenues for future treatment development
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