Mechanism Of Bradykinin Selectively Increasing The Permeability Of Blood-tumor Barrier

The blood-brain barrier(BBB)is a dynamic interface that controls the influx and eftlux of a wide variety of substances and consequently restricts the delivery of drugs into the central nervous system(CNS).There are two pathways of drugs being delivered into the tumor cells blood-tumor barrier(BTB):paracellular pathway and transcellular pathway.Studies show that small-dose bradykinin(BK)may selectively open the BTB,and no normal brain is involved.But the identified mechanism of BK to selectively open BTB is unclear.Although some observations by measuring transendothelial electrical resistance (TEER)of BTB model in vitro after using BK shows that the value of TEER decreases and TJ opens,so far,BK whether could open TJ still to have the controversy and the precise molecular mechanism of how TJ is opened by BK is not further illustrated.In addition,many researches reveal that BK increases permeability in malignant glioma through the transcellular pathway(the pinocytotic vesicles were increased).Classical transcellular pathway involving clathrin-coated pits,other ligand induced internalization pathways have been described that involves caveolae and other clathrin/caveolae independent mechanisms.The pathway of BK increasing the pinocytotic vesicles is unclear.3'5'-cyclic adenosine monophosphate(cAMP)is an important second messenger in the cell and participates in many singal pathways.Some studies indicate that the increase of cAMP leads to up-regulation of TJ associated proteins and an increase of TEER value,which results in intensified TJ.Moreover,cAMP regulates TJ by protien kinase A(PKA)-dependent or PKA-independent pathway.Whether cAMP/PKA signaling molecules are involved in the regulating process of BK opening TJ is unclear.This study is performed to determine whether BK opens the BTB by affecting tight junction(TJ)associated proteins zonula occluden-1(ZO-1),occludin,caludin-5 and cytoskeleton protein filamentous-actin(F-actin)and whether cAMP/PKA signaling molecules are involved in the regulating process.At the same time,to study whether caveolae is involved in the processing of BK opening the BTB in the same time.Methods1,Establishment of rat C6 brain tumor model and BTB model in vitro.2,In C6 brain glioma rats,the BTB permeability was determined by Evans blue (EB)assay,the state of TJ and the quantity of pinocytotic vesicles were determined by transmission electron microscopy(TEM)before and after BK infusion.3,Millipore electrical resistance system(Millicell-ERS)was used to detect the TEER of BTB model in vitro.4,Horseradish peroxidase(HRP)assay was used to measure the permeability of BTB model in vitro.5,Before and after BK admistering,immunohistochemistry and immunofluorescence assays were used to determine the distribution of TJ associated proteins ZO-1,occludin,claudin-5 and cytoskeleton protein F-actin in BTB model in vivo and in vitro and the distribution of caveolin-1 and caveolin-2 in C6 brain glioma rats.6,Before and after BK admistering,Western blot assessment was used to detect the proteins expression levels of TJ associated proteins ZO-1,occludin and claudin-5 in BTB model in vivo and in vitro and the levels of caveolin-1 and caveolin-2 in C6 brain glioma rats.7,Investigation the content of cAMP and the protein expression level of PKA catalytic subunit(PKAcs)in C6 brain glioma rats by radio-immunity and Western blot assay repectively,before and after BK infusion.Results1,C6 glioma rat model and BTB model in vitro were established successfully.2,In C6 glioma rats,the TJ was opened and the permeability of BTB was increased after BK infusion.TEER values were decreased and HRP permeability was increased in BTB in vitro.The above various measurements changed obviously at 15 min after BK administering.After that,they recovered gradually. 3,In C6 glioma rats,after administrating BK for 15 min,there was a significant decrease in TJ associated proteins ZO-1,occludin and claudin-5-like immunoreactivity and the immuno-staining of cytoskeleton protein F-actin on tumor capillaries compared with BK 0 min and control groups,From then on,it recovered gradually.4,In BTB model in vitro,the immunoreactivities of ZO-1,occludin and claudin-5 were attenuated and they were discontinuously distributed in the cellular boundaries and immunofluorescence were partly cytoplasmic after BK administering.The proteins expression levels of them were attenuated also.The immuno-staining of F-actin on cellular boundaries was attenuated and stress fiber was increased after administering.5,In C6 glioma rats,the protein expression level of caveolin- 1 was increased after BK infusion.It formed a peak at 15 min,then decreased and finally restored to the level of BK 0 min group.There was no significant difference of the expression level of caveolin-2 at the different time points after BK infusion.6,There was a significant decrease in expression level of cAMP and PKA respectively in C6 glioma rats.The lowest expression levels of them were appeared at 15 min after BK infusion.DiscussionOur results revealed that BK could open the tight junction and increase the pinocytotic vesicles,then increase the permeability of BTB.Compared with control group,the expression levels of TJ associated preteins ZO-1,occludin and claudin-5 were decreased significantly after BK administering and the lowest levels of them appear at 15 min.At the same time,the study also indicates that the distribution of TJ associated proteins and F-atin is altered.The expression level of caveolae structural protein caveolin-1 is significantly increased compared with the control after applying BK to BTB model in vivo.The peak is formed at 15 min after BK administering.The expression of caveolin-2 is not changed by BK inducing.Those results showed that BK could increase the permeability of BTB by both paracellular pathway and transcellular pathway.Small dose of BK leads to increasing permeability of BTB without affecting normal brain.Our assay of EB extravasation proved that BK selectively increased the permeability of BTB in experimental glioma rats and permeability of EB reached to the peak at 15 min after infusion BK.As far as the mechanism of BK action is concerned, most of researchers concentrate on the transcellular pathway of increasing pinocytotic vesicles.To summarize our TEM studies of microvessels,we observed that endothelial pinocytotic vesicular density was increased after BK infusion.This result proved the results of previous studies.Sanovich et al.found that BK agonist RMP-7 could transport drugs by opening TJ when they studied the pathway of its opening the BBB. Meanwhile,Easton et al.showed by experiments in vitro that BK decreased TEER of endothelial cells and opened TJ.These results are coincident with our results.Our TEM result revealed that TJ was opened after BK administration.Meanwhile,we measured the TEER of BTB model in vitro after BK administration and found BK decreased the TEER,the lowest level of TEER appeared at 15 min.The HRP flux assay revealed that BK induced an increase of HRP flux in a time-dependent manner and the peak of HRP flux at 15 min after BK administering also.Based on the results of those studies mentioned above we can conclude that BK could open the TJ and increase the pinocytotic vesicles.But the precise molecular mechanism is unclear.In order to make the mechanism of BK opening the TJ clear,the expression and distribution of ZO-1,occludin and claudin-5 are measured with Western blot and immunohistochemistry assays on BTB model not only in vivo but also in vitro at the same time.ZO-1 is the first identified protein of TJ proteins.It is a member of the MAGUK protein family(it is membrane associated and with guanylate kinase region or GUK domain).Increasing evidence indicates that the loss of ZO-1 could result in a disorganization of TJ.Occludin is the best-known integral membrane protein of TJ, which is believed to be directly involved in its barrier and fence functions.Claudin-5 is a major cell adhesion molecule of tight junctions in brain endothelial cells and that it may be directly involved in the establishment of the BBB and regulation of the permeability of BBB.Our results show that BK reduces the protein expression and changes the distribution of ZO-1,occludin and claudin-5,and the most obviously changes appeared at 15 min after infusion BK.The time point that the changes of ZO-1, occludin and claudin-5 reach the peak are the same as time points that EB,TEER and HRP reach the peak.Those indicate that BK could affect the tightness of TJ by down-regulating the expression of ZO-1,occludin and claudin-5.Recent researches imply the important roles of actin filaments in both structural support and functional influence on the organization of TJ complex,and of modulating permeability of BBB, subsequently.We observed the change of F-actin by fluorescence microscope;the result showed that an obvious depolymerization of F-actin during BK infusion and the change of F-actin was correlated with the opening of TJ.By a large number of researches,no matter in vivo or in vitro,it is proved that the interaction between TJ associated proteins and cytoskeleton protein maintains the integrity of TJ together. Consequently,we consider that BK might through affecting the action of F-actin to regulate the structure and function of TJ associated proteins and result in opening the tight junction.Classical transcellular pathway involving clathrin-coated pits,other ligand induced internalization pathways have been described that involves caveolae and other clathrin/caveolae independent mechanisms.Caveolin-1 is essential for the formation of caveolae and is a marker for caveolae.Many investigations reveal the change of the caveolin-1 expression level is associated with the increasing of the permeability of BBB and caveolin-2 collaborative caveolin-1 completed endocytosis function.We used immunohistochemistry;immunofluorescence and western blot assays to research the distribution and expression of caveolin-1 and caveolin-2 after BK administering.The results showed that the distribution was changed and the expression level was increased of caveolin-1 and the peak was appeared at 15 min.The changes were similar to the time of the increasing of pinocytotic vesicles.The distribution and expression of caveolin-2 was not involved.So we considered that BK could increase the permeability of BTB through caveolin-1 mediated transcellular pathway.All the results mentioned above indicate that BK could open TJ by affecting TJ associated proteins ZO-1,occludin and claudin-5,and cytoskeleton protein F-actin and could increase pinocytotic vesicles by increasing the expression of caveolin-1.For the first time we prove BK increases the permeability of BTB by both paracellular pathway and transcellular pathway at molecular biology level.Conclusions1,BK could open TJ,raise the quantity of pinocytotic vesicles and increase the permeability of BTB selectively.2,The expression levels of TJ associated protein ZO-1,occludin,claudin-5 and cytoskeletal protein F-actin were decreased by BK,which might play an important role in the opening of TJ by BK induction.3,BK could promote the expression of caveolin-1 in C6 brain glioma rats.The up-regulating of caveolin-1 might be associated with the increasing of pinocytotic vesicles by BK induction.4,BK could inhibit the expression of cAMP and PKAcs,which suggested cAMP/PKA signaling pathway might be involved in regulating of BK opening TJ.5,BK might increase the permeability of BTB selectively by both transcellular pathway and paracellular pathway.