Expression And Roles Of Vascular Endothelial Growth Factor-C In Mesial Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy(MTLE) is a frequent form of focal intractable epilepsy in adults. Recurrent seizures often promote the disruption of blood-brain barrier(BBB) and brain inflammation, which, in turn, exacerbate seizures per se. Accumulating data from experimental epilepsy models and resected human brain tissues of intractable epilepsy indicate the involvement of innate immune system activation and consequent brain inflammation in TLE. And BBB disruption has also been found to be associated epilepsy. Numerous experimental animal and human studies demonstrated that BBB disruption could be caused by seizures. And BBB opening in the chronic epileptic phase of TLE leads to a persistent increase in the number of seizures, indicating that BBB disruption could also contribute to the progression of epilepsy or aggravate seizure activity.Most MTLE patients become drug-resistant and have to undergo surgical resection of the epileptic foci. The specific mechanism underlying the epileptogenesis of MTLE is still unknown but studies performed on surgical tissues of MTLE and relevant rodent models suggest that many growth factors have participated in the initiation and development of MTLE, such as vascular endothelial growth factor(VEGF), brain-derived neurotrophic factor and nerve growth factor.Vascular endothelial growth factor(VEGF), also known as VEGF-A, is regarded as a key regulator of angiogenesis and vascular permeability. Previous study also suggest that VEGF-A has neurotrophic and neuroprotective properties. The role of VEGF-A in epileptogenesis has been widely studied. In addition to VEGF-A, the VEGF family consists of six other polypeptide growth factors, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor. VEGF-C, exhibiting ~30% homology to VEGF-A, exerts its function through two high affinity tyrosine kinases, VEGFR-2 and VEGFR-3. VEGF-C was initially identified as a major regulator of lymphangiogenesis, as well as a potent mediator of angiogenesis in embryo. However, increasing evidence indicates that VEGF-C is also distributed in the brain and play important roles in physiological and pathophysiological states by coupling two high affinity tyrosine kinases, VEGFR-2 and VEGFR-3. It has been demonstrated that VEGF-C acts on neural progenitors as a trophic factor and regulates neurogenesis in both developing and adult brains. In addition, VEGF-C and its receptors have been indicated to participate in glial reaction after some brain insults. Shin et.al reported that VEGF-C/VEGFR-3 is induced in reactive astrocytes and activated microglia in rats with ischemic stroke. A similar expression pattern for VEGF-C/VEGFR-3 has been found in the striatum of adult normal rats that received bone marrow-derived human mesenchymal stem cell. And administration of VEGF-C into intact brain activates astroglia and microglia. Our previous study has shown increased expression of VEGF-C and its receptors, VEGFR-2 and VEGFR-3, in reactive astrocytes in epilepsy-associated tuberous sclerosis complex Notably, our recent data revealed that VEGF-C and its receptors, VEGFR-2, VEGFR-3, were up-regulated in cortical tubers of tuberous sclerosis complex(TSC), which has been proven to be a pivotal cause of pediatric intractable epilepsy. VEGF-C primarily acts on lymphagiogenesis, but previous studies have also addressed its role in angiogenesis. It was reported that VEGF-C induces angiogenesis in the striatum in a rat model of Parkinson’s disease. Moreover, intracerebral injection of VEGF-C could disrupt blood-brain barrier in intact rat brain.However, the association between VEGF-C and human MTLE has not yet been described.In the present study, we aim to investigate the levels and expression pattern of VEGF-C and its receptors, VEGFR-2 and VEGFR-3, in surgically resected brain tissues from patients with MTLE and in pilocarpine-induced mouse MTLE modelsto elucidate the contribution of VEGF-C and its receptors to the epileptogenesis of human MTLE.And using video electroencephalogram(EEG), we examined the effect of VEGF-C on the seizure activities in animal models. We also explored the potential roles of VEGF-C in regulating the levels of albumin, a biomaker of the BBB disruption, and several proinflammation cytokines involved in epileptogenesis of MTLE, as well as the expression of phospho-S6(p-S6), the indicator of activation of mammalian target of rapamycin(mTOR) pathway. Since VEGF-C could increase the release of IL-17 in the mouse MTLE models, we investigated its expression and cellular distributionin patients with MTLE. We obtained the following results:1. Real-time quantitative polymerase chain reaction(qPCR), western blotand enzyme-linked immune response(ELISA)results revealed up-regulated levels of VEGF-C, VEGFR-2 and VEGFR-3 in the MTLE group compared to the control groups.2. Immunohistochemistry and double-labeled immunofluorescence showed that VEGF-C was highly expressed in neurons, astrocytes, including reactive astrocytes, and vascular endothelial cells, VEGFR-2 was expressed at a high level in reactive astrocytes and vascular endothelial cells, but not in neurons, whereas VEGFR-3 was only overexpressed in reactive astrocytes.3. The immunoreactive protein levels of VEGF-C, VEGFR-2 and VEGFR-3 were increased in pilocarpine-induced mouse MTLE models at 3 hour(h), 1 week(w) and 3 w.4. VEGF-C increased the spike frequency and the number of seizures in MTLE mouse models at 3 h, 1 w and 3 w. And the effect was alleviated by antagonist of VEGFR2 or VEGFR3.5. VEGF-C up-regulated the levels of albumin, the production of IL-1β,IL-6,IL-17, TNF-α, and the expression of p-S6 in the mouse models of MTLE at 3 h, 1 w and 3 w. Blocking of VEGFR-2 or VEGFR-3 abolished the effect, blocking VEGFR-3 did not alleviate the effect of albumin.6. IL-17 and IL-17 receptor(IL-17R) were clearly upregulated in MTLE at both mRNA and protein levels, compared with control specimens.7. Immunostaining indicated that IL-17 and IL-17 R were highly expressed in neurons, astrocytes, microglia, and endothelial cells of blood vessels.Taken together, these findings suggest that VEGF-C and its receptors, VEGFR-2 and VEGFR-3, may contribute to the epileptogenesis of MTLE.