Fluoxetine induces vascular endothelial growth factor/Netrin over‐expression via the mediation of hypoxia‐inducible factor 1‐alpha in SH‐SY5Y cells

Abstract

Fluoxetine has become one of the most promising drugs for improving clinical outcome in patients with cerebral infarction. Although the clinical efficacy of fluoxetine has been preliminarily demonstrated, its mechanism remains unclear. Hypoxiainducible factor 1-alpha (HIF-1a) is upstream to Netrin and vascular endothelial growth factor (VEGF), and under hypoxia conditions it may induce expression of Netrin-1 and VEGF in vascular endothelial cells. We sought to explore whether it can regulate their expression in hypoxia and mediate the effect of fluoxetine in hypoxia. In this study, the effect of hypoxia on the expression of VEGF and Netrin was observed in vitro by realtime PCR and western blotting in SH-SY5Y cells; the binding sites of HIF-1a in VEGF and Netrin gene promoters were identified by luciferase reporter; the effect of fluoxetine on binding of HIF-1a with Netrin and VEGF promoters in hypoxia was observed by Chromatin Immunoprecipitation (ChIP) Assay. We prove that HIF-1a regulates transcription of both VEGF and Netrin, and that in hypoxia fluoxetine up-regulates VEGF and Netrin expression via mediation of HIF-1a that binds to hypoxia-response element sites of VEGF and Netrin promoters. Our study indicates that HIF-1a may play an important role in the treatment of cerebral infarction through mediating the recovery of neurological function induced by fluoxetine, which provides theoretical basis for the development of gene therapeutic drugs targeting HIF-1a. Keywords: fluoxetine, hypoxia-inducible factor 1-alpha, ischemic infarction, netrin, vascular endothelial growth factor. Cerebral infarction is a clinically common disorder of nerve function impairment for which the most effective treatment is thrombolytic therapy at acute stage. As a key part of clinical management, the recovery treatment for damaged neurological function during infarction convalescence stage still lacks drugs with demonstrated efficacy. Among the possible drugs, monoamines are supposed to be the most promising ones. A number of animal experiments suggest that selective serotonin reuptake inhibitors (SSRIs) (Liepert 2008), which bind to 5-hydroxytryptophamine (5-HT) transporter and inhibit 5-HT reuptake thus increasing 5-HT levels at synaptic cleft, may protect injured ischemic brain tissue and promote regeneration of hippocampal neurons (Li et al. 2009;Lim et al. 2009). Some small studies suggest that these drugs may improve motor function (Loubinoux et al. 2002). Among SSRIs, fluoxetine is the one most extensively used in management of various depression symptoms. Important progress was made in 2011 with a randomized, placebocontrolled trial, Fluoxetine for Motor Recovery after Acute Received October 22, 2015; revised manuscript received December 2, 2015; accepted December 21, 2015.Address correspondence and reprint requests to Qiaoshu Wang, MD, PhD, Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 100 Haining Road, Shanghai 200080, China. E-mail: qwang624@139.com 1 These...

Use of dual-flow bioreactor to develop a simplified model of nervous-cardiovascular systems crosstalk: A preliminary assessment

“…The release of VEGF in the medium by SH-SY5Y cells may be interpreted as a reaction to hypoxia, this is in accordance with previously published data showing that oxygen deprivation caused VEGF production in SH-SY5Y at both mRNA and protein level, and with the evidence of a correlation between AngII and hypoxic conditions [47,48,[52][53][54][55]. SH-SY5Y cells have AngII type 1 receptor localized in the membrane [56], and when treated with AngII they displayed a stressed behaviour with subsequent upregulation of factors responsible for VEGF-mediated angiogenesis [8,57].…”

Netrin-1 Stimulates Migration of Neogenin Expressing Aggressive Melanoma Cells

“…Western blot analysis was used to determine baseline expression of Netrin-1 and Neogenin in the melanoma cell lines C8161, Sk-Mel28, UACC1273, and WM1552C. The neuroblastoma cell line SH-SY5Y and the human normal keratinocyte cell line HEKn were used, respectively, as high and poor cellular expression controls for Netrin-1 [ 26 , 27 ]. WB results show variable expression of Netrin-1 in the different melanoma cell lines ( Figure 1 A).…”

HIF‑1α in cerebral ischemia (Review)

“…Therefore, it is necessary to study further feasible and effective treatment options. A number of studies (119)(120)(121)(122) have demonstrated that certain natural compounds, chemical drugs and traditional Chinese medicine compounds may alleviate cerebral ischemic injury through HIF-1α. A previous study found that certain compounds and traditional Chinese medicines serve a role in angiogenesis and vascular protection through the HIF-1α/VEGF signaling pathway, thereby protecting against cerebral ischemic injury (123).…”

“…Astragaloside IV activates the HIF-1α/VEGF/Notch signaling pathway through miRNA-210 to promote angiogenesis ( 121 ). Fluoxetine induces a cascade of events leading to the upregulation of the expression of HIF-1α-Netrin/VEGF protein, promotes angiogenesis after ischemic stroke and improves long-term functional recovery after ischemic stroke ( 122 , 123 ). Racemic dl-3-n-butylphthalide treatment could also promote functional recovery after focal transient cerebral ischemia, and this recovery and dl-NBP may upregulate the expression of HIF-1α-VEGF and Notch-Dll4, and then affect the integrity of white matter, the number of capillaries and the expression of tight junction protein occludin ( 124 ).…”