In Vitro Study Of The Mechanism Of Bradykinin Selective Opening The Blood Brain Barrier

Glioma is a common malignant brain tumor, which has a poor clinical prognosis. Chemical therapy remains an efficient treatment to prolong the life of malignant tumor today, but unfortunately, brain tumor capillaries constitute the blood - tumor barrier ( BTB) , which despite being somewhat more leaky' than the BBB, also limits delivery of antitumor agents to the tumor. Some studies showed that if the drug concentration in the tumor tissue be elevated by two fold, the efficacy to kill the tumor cells could increase by 10 fold. So, efficient delivery of drugs to tumor cells is crucial for effective chemotherapy of glioma. It was found that, in contrast to large dose of BK that could breakdown the whole BBB, small dose of BK could selectively open the blood tumor barrier thus enhance the delivery of some large particles which normally impeded by the blood tumor barriers. In other words, small dose of bradykinin could deliver chemotherapy drug to tumor tissues selectively. The selective modulation of blood tumor barrier provides a promising method for efficient chemotherapy. Pharmacological and molecular biological studies have revealed the existence of two major subtypes of bradykinin receptors, B₁ and the G - protein coupled B₂, which mediate the effects of bradykinin in different cell types. In vivo study showed that the selective increases in BTB permeability induced by intracarotid infusion of low doses of bradykinin are mediated solely by B₂ receptors because such permeability increase can be significantly inhibited by the B₂ receptor antagonists, HOE 140 and D - Arg, but not by the B₁ receptor antagonist.In our previous animal study, we have shown that the B₂ receptor expressed mainly in the tumor cells while the normal and tumor capillary do not express the B₂ receptor.But it still could not clarify why small dose of BK could open the BTB, and large dose of BK breaks down the whole BBB. Based on the primary culture of rat astrocyte and BMEC, after the study of intracellular calcium change when different doses of BK were applied and Western blot analysis of B2 receptor, now we could give a much further explanation to above questions.Materials and Methodslx Primary culture of Brain Microvascular Endothelial Cells and Astrocyte: Wistar rat, aged 1—2 week, were used as the source of microvessels. Animals were sacrificed by C02 inhalation, immediately after death, craniotomy was performed, and the entire cerebrum dissected free of meninges. The protocol to establish BMEC consisted of following three main phases: (1) the isolation of a microvessel fraction, (2) digestion of the microvessels. A crude fraction of microvessels was isolated from cortical tissue by centrifugation through 15 ml of 18% (wt/vol) dextran solution and centrifuged at 10,000 g for 10 min, and then they were placed in D - Hank's solution containing lmg/ml collagenase. When the microvessels appear "beads on a string" , fed with fresh, complete culture media. Cultures were passaged only one before analysis and were identified by VIII factor immunohistochemistry. The " foamy layer" was resuspended by gentle aspiration to isolate the astrocyte and was identified by GFAP immunohistochemistry.2 % Measurement of intracellular Ca + by Fluo - 3 immunofluorescence: C6 glioma cell, Astrocyte and BMEC were cultured on a circular glass when cells grew and covered the glass. DMEM containing Fluo - 3/AM (5|xmol/ml) was added to incubate at 37°C for 30 min;The artificial cerebro-spinal fluid (ACSF) : (126 mmol/ml NaCl, 3. 75 mmol/ml KC1, 2 mmol/ml CaCl2f 1 mmol/ml MgCl2f 26 mmol/ml NaHC03, 1.25 mmol/ml KH2PO4, 10 mmol/ml glucose, pH 7.4) with distilled water (1:1) was used to wash extracellular free Fluo -3/AM and incubated for another 30 min. When loaded over and washed trice with ACSF, the loaded cells were suspended in lml ACSF solution and receive a test with a fluorescence spectrophotometer. Raster (EX) 5nm, radiate raster (EM) 10 nm were excited at a middle scan speed (32 mm/ min) , excitation light scan ranged 490 nm, emission light scan ranged 520 nm. Fluorescence intensity was positive correlated with the free intralcelluar Calculation of [ Ca + ] i .3 N Western blot of B2 receptors;Protein homogenates were prepared from BMEC, C6 and Astrocyte cells by rapidly homogenized by 10 volumes of lysis buffer ( 1% SDS, lmM Sodium Van-adate, lOmM Tris PH 7.4). Samples were centrifuged at 20,000 x g for 15min and the protein concentration of soluble materials was determined by the BCA method, then they were appropriately diluted to equal concentrations of total protein. Control protein lysate for B2 receptor was provided by Transduction Laboratories. Protein lysates were fractionated on 12% SDS - polyacrylamide gels and Western blots prepared by electroblotting proteins to polyvinylidene difluoride (PVDF) membranes. Western blots were blocked for lh by incubation in 10% milk powder dissolved in PBS, and washed with PBS. These blots were incubated with anti - B2 receptor antibody for lh, washed trice with PBS, and then incubated with peroxidase - conjugated rabbit anti - mouse immunoglobulin for lh. B2 receptor bands on these immunoblots were visualized using the enhanced chemiluminescence (ECL) kit and by exposing photographic films to the immunoblots for 2 min.ResultslxWe used homogenization, dextran density centrifugation to isolate brain microvessels, which digested by collagenase were cultured in 37 X! , 5% CO2 incubator. Immunocytochemistry demonstrated more than 95% culture cell are positive to VIII factor protein, that indicates the culture cells are vascular endothelial cell. Tight junction protein - occludin is positive around the border of the BMECs, which indicated that the BMECs have BBB characteristics.2xWhen small dose of BK (final concentrations: 10"5 M) was ap-plied, the intracellular calcium in C6 elevated suddenly, and form a peak after 30sec, then it decreased and finally restored to a normal calcium level. Contrast to C6 glioma cell, astrocyte remains unresponsive to small dose of BK (10⁵ M) , but when large dose of BK (final concentrations: 10 M) was applied, its intracellular calcium increased instantly and form a peak at 10s. No matter large or small dose of BK was applied, the intracellular calcium in BMECs is not changed, which indicated that BK has no direct influence on the BMECs. Selective B2 receptor antagonist D - Arg blocked BK induced intracellular calcium response in C6 glioma cell.3 x Western blot analyses with anti - B2 receptor monoclonal antibody revealed a 42k Da protein, identifiable as a single band in lysates of cultured cells. The result of the western blot of B2 receptors demonstrated that the B2 receptor is not located in the BMEC. The astrocyte express small amount of B2 receptor while the B2 receptor is abundant in the C6 glioma cell line. The IDV (Integrated Density Value) of three lines of cell is 5000. 12±1110. 21 (n=2) , 18480.88*4119.86 (n=2) , 63032. 13*2802. 71 ( n = 2) respectively, and there is an apparent statistical difference between each group.Conclusionsl%Homogenization, dextran density centrifugation, enzyme digestion are a series of stable method to isolate and culture of the primary brain microvascular endothelial cell.2%The direct target cells of bradykinin are astrocyte and C6 tumor cell, relying on some intercellular messenger, bradykinin could modulate the permeability enhancement of the BMEC.3 xThe different expression of B2 receptors in astrocyte and glioma cell contributes to the selectivity of small dose of BK' s selective modulation of the bloodtumor barrier.