| Cerebrovascular diseases and brain neurodegenerative diseases are becoming the leading captured items in the domain of medicine, which have the characteristic of high incidence rate, high crippling rate, and high recurring rate. In recent years, through the repeatedly clinical observation and practice, it is thought that these diseases belong to the category of'collateral disease'in Chinese medicine, and 'toxin hurts brain collaterals' is the key point in its pathogenesis. It is also realized that brain microvascular endothelial cells (BEC) are not only the substance exchange barrier of blood and tissue, but also have important incretion function. Our researches pay more attention to BEC rather than neurons, which is a meaningful probe to guide Traditional Chinese Medicine mordernation in brain diseases.The major goal of this study was to assess the molecular mechanism of brain microvascular endothelial cells affects cerebral micro-environment.The experiments are divided into three parts:First:We employed transgenic (Tg) mice model. Tg DNPPET mice were crossed with Tg KAPP animals to produce Tg KAPP/DNPPET animals, as well as single Tgs (Tg KAPP, Tg DNPPET) and nonTg littermates. We measured MMP-2 activity, VCAM-1 and ET-1 expression and involved signal transduction in four groups.Second:By culture mice brain microvascular endothelial cells, we observed Receptor for advanced glycation end products (RAGE) mediated MMP-2 expression in Aβinvolved BEC and signal transduction.Third:We examined the expression of Placental growth factor (PlGF) in cerebral ischemia, utilizing a permanent middle cerebral artery occlusion (MCAO) model in the rat. The effects of PlGF upon neuronal vascular endothelial growth factor receptor-1 (VEGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2) expression were also examined.Research Results:1. By immunohistochemical analysis with 3D6 antibody in four genotypes of mice, there was a significant decrease in the extent of total Aβdeposition in the cortex in Tg KAPP/DNPPET mice compared with Tg KAPP mice. In contrast to the parenchymal, a more striking decrease in the amount of cerebral vascular A(3 was observed in the Tg KAPP/DNPPET mice.2. MMP-2 activity, VCAM-1 and ET-1 expression have been shown to be elevated in Tg KAPP mice compared with non Tg mice, however, we found markedly decreased MMP-2 activity and protein levels of VCAM-1 and ET-1 in Tg KAPP/DNPPET mice compared with Tg KAPP mice.3. Levels of phosphorylated JNK and ERK 1/2 were significantly increased in brain extracts of KAPP and KAPP/DNPPET mice, as compared to nonTg mice, while KAPP/DNPPET mice revealed significantly less phosphorylation of JNK and ERK 1/2, as compared with KAPP mice.4. Compared with control group, MMP-2 expressions were significantly increased in Aβ-induced BEC. Pretreatment with ERK, JNK MAP kinase inhibitor PD98059, SP600125 resulted in a highly significant reduction in Aβ-induced upregulation of MMP-2.5. Introduction of anti-RAGE to block RAGE exerted similar suppressive effects on Aβ-stimulated upregulation of MMP-2.6. PlGF immunoreactivity cells were increased in cells of the mesenchyma and in the vascular interstitium in the MCAO group compared with the sham group, and its peak time of expression is at 72h.7. In normal control neurons, VEGFR-2 cellular content was increased by the addition of PlGF. In comparison with the normal control neurons, the VEGFR-2 expression of OGD-treated neurons was significantly increased (p<0.01), confirming the results of the western blotting. VEGFR-2 cellular content in OGD neurons was also significantly increased by the addition of PlGF to OGD-treated neurons (p<0.01). Conclusions are made as follows:1. By transgenic mice model TgDN-RAGE, it was shown that DN-RAGE can affect deposition of Aβ, particularly on the cerebral vasculature. It also had influence on the levels of total brain Aβ, although there was not a significant different, notably, elevated the levels of plasma Aβ. It was suggested that RAGE was involved with facilitating diffuse Aβdeposition in the parenchyma, and also in the cerebral vasculature.2. DN-RAGE displayed decreased activity of MMP-2 and VCAM-1, ET-1 expression. Detailed mechanisms linked activation of JNK MAP kinase and ERK1/2. These data provided support for the concept that RAGE functions as a signaling receptor, rather than solely as a site tethering ligands to the cell surface and also suggested that DN-RAGE can weaken vascular inflammatory stress in Alzheimer disease.3. In cultured BEC, our data suggested that RAGE mediated MMP-2 expression in Aβ-induced endothelial cells, and ERK, JNK MAP kinase signal transduction involved. In all, our experiments supported the possibility that blockade of vessel RAGE might have protective effects, especially with respect to attenuate the pathogenesis of vascular inflammation in AD.4. It was shown that PlGF was expressed in rat brain and this expression was found mainly in brain microvascular endothelial cells. PlGF expression was significantly increased after cerebral ischemia injury and this increase has a neuroprotective effect in response to OGD injured neurons. We also found that VEGFR-2 signaling may play a role in PlGF-mediated neuroprotection. PlGF is, therefore, a promising target for therapeutic intervention in ischemic injury. |
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