The Roles And Mechanisms Of VEGF-C In Malformations Of Cortical Development

Malformations of cortical development (MCD) represents a well-recognized cause of intractable epilepsy, with a poor response to the currently available anti-epileptic drugs (AEDs). With the development of high-resolution imageology, the detection rate of MCD is continuously rising during the past decade in clinic.It has been demonstrated that approximately 40 percent of adult patients and more than 80 percent of pediatric with intractable epilepsy are associated with MCD.Thus, the pathogenesis of MCD has became a central concern in the field of intractable epilepsy. MCD encompasses a large spectrum of disorders related to abnormal cortical development, including tuberous sclerosis complex (TSC), focal cortical dysplasia (FCD), microgyria, congenital agyria and Sturge-Weber syndrome. Among these subsets, two of the most common components are TSC and FCD. Despite the fact that these subsets with varied genetic etiologies, anatomic abnormalities, and clinical manifestations, they often share similar histological features such as disorganization or lack of the normal six-layered cortical lamination structure, and the occurrence of malformed neurons (MNs) known as dysplastic neurons (DNs), giant neurons (GNs),balloon cells (BCs) and giant cells (GCs). Mounting clinical evidence has revealed that epilepsy readily occurs in patients with MNs, and appears drug resistance in the early stage, suggesting the dominant role of MNs in epileptogenesis of MCD. Until now, the pathogenesis of MNs remains unclear. A more popular view is that MNs is a malformation due to abnormal migration,mature and cell death during embryonic brain development. Moreover, recent pathological studies on surgical or autoptic human tissue suggest a role for perinatal and early postnatal brain injury in the formation of MNs. In spite of the fact that there are many reports on the subject of MCD, the exact mechanism of this disease has not yet been elucidated.VEGF-C,a well-recognized regulator of lymphangiogenesis, binds to and activates the specific receptors VEGFR-2 and VEGFR-3, and the affinity of VEGFR-3 is much more than that of VEGFR-2. Previous studies show VEGF-C and VEGFR-3 are restrictly expresssed in lymphatic endothelial cells, and VEGFR-2 is expressed in both vascular endothelial cells and lymphatic endothelial cells.Due to the deficiency of the lymphatic system in the brain, few studies have focused on the role of VEGF-C in the CNS. Until to 2006, an important study demonstrats that VEGF-C and VEGFR-3 are present in the neural progenitor cells of Xenopus laevis and mouse embryos. Furthermore, a lack of VEGF-C results in a severe defect in the proliferation of neuronal progenitor cells expressing VEGFR-3, suggesting that VEGF-C is a trophic factor in neuronal progenitor cells in the vertebrate brain. Recently, several studies revealed VEGF-C mediate multiple effects in CNS. For example,activation of the VEGF-C/VEGFR-3 signaling pathway was shown to mediate proliferation and chemotaxis in glial precursor cells. In addition, after a short period of brain ischemia, the innermost layer of granular cells in the hippocampal dental gyrus displayed increased levels of VEGFR-3 expression, these VEGFR-3 immunoreactive cells are also expressed PSA-NCAM, this might be related to neurogenesis in the hippocampus after cerebral ischemia. A recent study showed that the expression of VEGFR-3 increased in subventricular zone, and most of VEGFR-3 positive cells were neural progenitors, were highly proliferative. To our knowledge, there have been no reports about the roles of VEGF-C in in seizure disorders, especially those induced by MCD. In our preliminary experiment,we found that VEGF-C mRNA exhibited high levels in MCD. However, the detailed expression pattern of VEGF-C and it receptors in MCD, and the role of VEGF-C signal system in the epileptogenesis of MCD are unclear.To address these concerns:First,we identified the distribution of VEGF-C system in MCD from patients with medically intractable epilepsy by means of RT-PCR, western blot and immunohistochemistry. Second,we tested the expression and activation of signaling molecule involved in VEGF-C pathway. Third, we investigated the expression and cellular distribution of VEGF-D,which is highly homologous with VEGF-C, in MCD. Fourth, to reveal the role of VEGF-C in the epileptogenesis of MCD, a MCD rat model was employed, and we investigated the effect of VEGF-C on NMDA receptor(NMDAR)of MNs by using whole-cell patch-clamp recordings technique on brain slice of MCD rat model. The results show as following: Ⅰ. Expression and cellular distribution of VEGF-C and VEGF receptors 2 and 3 in MCD1. RT-PCR and western blot analysis showed VEGF-C and its receptors mRNA and protein expression in total homogenates of normal control cortex (CTX); immunohistochemical experiments revealed immunostaining was mainly observed in pyramidal neurons.2. There was a statistically significant increase of VEGF-C, VEGFR-2 and VEGFR-3 mRNA and protein levels in cortical tubers of TSC, in comparison with CTX. The immunostaining results demonstrated that the high-level expression of protein, including VEGF-C and its receptors, was mainly localized within GCs, DNs, and reactive astrocytes. The results of double-labeling immunofluorescence showed:①the co-localization of VEGF-C immunostaining with the neuronal marker NF-200 in majority of DNs and GCs. Intriguingly, we could not detect any co-immunostaining of VEGF-C with another neuronal marker, NeuN, in both DNs and GCs in all cases. Also, GFAP and CD68 immunostaining was not detected in any VEGF-C-positive DNs and GCs.②VEGFR-2 and VEGFR-3 shared a similar cellular distribution pattern in TSC, VEGFR-2 (VEGFR-3) immunoreactivity co-localized with NF-200 in the majority of DNs,the frequency of VEGFR-2 (VEGFR-3) and NF-200 co-expression in GCs was less than that in DNs. VEGFR-2 (VEGFR-3) and GFAP co-immunolabeling was not detected in any DNs and GCs, although prominent co-labeling of reactive astrocytes was noted in all of the TSC cases examined.3. We found an obvious increase of VEGF-C and it receptors mRNA and protein expression in FCD specimens compared with CTX. The high-level expression of protein, including VEGF-C and its receptors, was mainly localized within DNs,GNs,GCs and reactive astrocytes. The results of double-labeling immunofluorescence showed:①GFAP and VEGF-C co-immunolabeling was not detected in any MNs. Interestingly,we detected co-immunostaining of VEGF-C with NeuN, which is usually recognized as a marker of mature neurons, in MNs.②VEGFR-3 and GFAP/NF-200 co-immunolabeling was not observed in any MNs,suggesting a complex phenotype of MNs.Ⅱ. The expression and activation of signaling molecule involved in VEGF-C pathway 1. In comparison with CTX, RT-PCR analysis showed a increase of Akt mRNA levels in MCD specimens. Immunoblots revealed a upward trend in the three types of Akt subsets and the most obvious change was observed in the expression of Akt1.These findings suggested the Akt pathway involved in the pathogenesis of MCD.2. A similar expression levels of Bad was detected in both CTX and MCD specimens, but we detected a significant increase of phosphorylated Bad (P-Bad) in MCD.3. We observed representative immunoblot double-bands of ERK1/2 (42 kDa and 44 kDa) in homogenates from MCD and CTX, and the ERK1/2 protein level in MCD was much high than that in CTX.Ⅲ. Expression and cellular distribution of VEGF-D in MCD1. We detected the VEGF-D mRNA and protein expression in total homogenates of CTX; immunohistochemical results revealed a weak immunostaining was restricted to pyramidal neurons.2. VEGF-D mRNA and protein levels were significantly increased in MCD compared with CTX. The immunostaining results demonstrated:①In TSC specimens, the high-level expression of VEGF-D protein was mainly localized within most of DNs,GCs, and reactive astrocytes; confocal images showed no co-localization of VEGF-D with NeuN, NF-200, GFAP and CD68 in MNs;②In FCD subtype, we found the VEGF-D was highly expressed in DNs,GNs,BCs and reactive astrocytes; the results of double-labeling showed no co-localization of VEGF-D with NF-200 and GFAP in these MNs;V.The modulatory effects of VEGF-C on NMDAR function in MNs from MCD rats model.1. We employed a well-established MCD rat model by in utero irradiation, and In vitro brain slices were obtained from these rats.The results of patch clamp recording showed:①With step-wise depolarization, MNs of MCD responded initially with action potentials (APs),but the shape of APs varied, the spike frequence adaption (SFA) was damaged,even disappeared,suggesting a immature characteristic in electrophysiology.②We successfully observed evoked-EPSC in MNs, and the amplitude and area of evoked-EPSC of MNs were clearly increased,suggesting a complex response comprising multiple inward currents;③The current ratio of NMDAR/AMPAR was increased in MNs compared with pyramidal neurons from normal control, mainly induced by the increase of NMDAR-mediated current, revealing a upregulation of the function of NMDAR.2. The immunostaining results showed there was a significant increase of VEGF-C and VEGFR-2 expression in MNs from MCD rats; the expression level of VEGFR-3 was also statistically increased.3. 100μmol/L NMDA could evoke the inward. After treatment with 100 ng/mL VEGF-C,100μmol/L NMDA caused a pronounced increase of transmembrane current in MNs, the data indicated a synergistic action between VEGF-C and NMDAR in MNs from MCD rats. We also found that Ki8751 (1 nmol/L), a VEGFR-2 inhibitor, abolished the upregulative effects of VEGF-C on NMDA receptor channel, this finding illuminated that the potentiation effects of VEGF-C on NMDAR is mediated by VEGFR-2.In summary, in the present study, we found an obvious increase of VEGF-C and it receptors mRNA and protein expression in MCD specimens compared with the control sample. The high-level expression of protein, including VEGF-C and its receptors, was mainly localized within MNs(DNs,GNs,GCs and BCs). Subsequently, we observed the activation of signaling molecule Akt, Bad, and ERK in MCD, which suggested the VEGF-C miediated anti-apoptotic mechanism might involve in the pathogenesis of MCD. Moreover, our data showed that NMDA receptor-mediated current is markedly potentiated by VEGF-C in the MNs from MCD rats, which suggests that a synergistic action between VEGF-C and glutamate systems exists in MCD. Altogether, these findings illuminate the VEGF-C system involves in the pathogenesis of MCD.