| ObjectiveBlood-brain barrier (BBB) is specialized to limit brain drug delivery. How to deliver macromolecular drugs across BBB to cure central nervous system diseases is a crucial problem.Recently, research showed endogenous receptors in brain microvascular endothelial cells mediated drug transport may be the most effective methods of delivering drug into brain. Researchers coupled drugs or gene transfer vectors to some monoclonal antibodies of endogeneous receptor, like transferrin-or insulin receptor, which showed high transport efficiency. These monoclonal antibodies competitive inhibit endogeneous ligands as transferrin and insulin to obstruct brain entry of essential compounds or drugs. New research showed cross-reacting material 197 (CRM197), the non-toxin mutant of diphtheria toxin which binding to diphtheria toxin receptor (DTR) localized in brain microvascular endothelial cells could transport macromolecular substance across the BBB in vitro. CRM197 had been used as a carrier protein for capsular polysaccharide antigen or vaccine in clinic abroad. Since no endogeneous competitive ligands exist in our body, CRM197 may become a new and more effective vector to deliver the drugs into the brain.There are two pathways of drugs being delivered across BBB:paracellular pathway and transcellular pathway. Caveolae-dependent transcellular pathway play an important role in the transcellular pathway. Caveolin-1 is identified as the main caveolae protein family member influencing the structural and functional properties of caveolae. Recent studies showed FOXO (forkhead box O) transcription factors could regulate caveolin-1 expression via direct interaction with a caveolin-1 promoter. FOXO transcription factors are under direct control of the PI3K (phospatidyl-3-inositol-kinase)/Akt (v-akt routine thymoma viral oncogene homolog) signaling cascade. FOXOs are phosphorylated by activated Akt and subsequent transcriptional inactivation. CRM197 bind to DTR and inhibit its shearing, which could decrease the product of heparin-binding epidermal growth factor (HB-EGF), attenuate PI3K/Akt pathway activated by HB-EGF, regulate the activity of FOXO transcription factors and up-regulate caveolin-1 expressing levels.The transcellular pathway mediate drug delivering across BBB is through opening the tight junction to increase the permeability of BBB by regulating the tight junction associated proteins like zonula occluden-1 (ZO-1), occludin, claudin-5 and adhesion proteins. No reports has been found about the pathway mediated the macromolecular substances across the BBB by CRM197.This research will elucidate the effects of CRM197 on the permeability of BBB in vitro and vivo and the mechanisms that CRM197 mediate the transport by which pathway. The function of PI3K/Akt/FOXO1A pathway on the regulation of caveolin-1 is also investigated.Methods1. Establishment of BBB model in vitro and prepare CRM197-HRP (horseradish peroxidase) and BSA-HRP conjugates.2. The transport effects of CRM197 were assayed by association experiment and transcytosis experiment using CRM197-HRP conjugate and human cerebral microvascular endothelial cells (hCMEC/D3).3. Millipore electrical resistance system (Millicell-ERS) was used to detect the TEER (trans epithelial electric resistance) of BBB model in vitro after treatment of CRM197.4. Evans blue (EB) was used to assay the changes of BBB permeability after administration of CRM197 in guinea pigs; transmission electron microscope observed the effects of CRM197 on the ultrastructure of brain microvascular endothelial cells.5. Before and after the treatment of CRM197, immunohistochemistry and immunofluorescence assays were used to determine the distribution and expression of p-Akt, p-FOXO1A and tight junction associated protein ZO-1 in hCMEC/D3 cells and the distribution and expression of caveolin-1 in brain microvascular endothelial cells in guinea pigs.6. Before and after treatment of CRM197, RT-PCR assay was used to detect the mRNA expressing level in hCMEC/D3 cells; western blot assessment was used to detect the proteins expression levels of caveolin-1, tight junction associated proteins ZO-1, occludin and claudin-5 and the activity of PI3K/Akt/FOXO1A pathway in hCMEC/D3 cells or in brain microvascular endothelial cells in guinea pigs.Results1. The BBB model in vitro were established successfully and obtained the CRM197-HRP and BSA-HRP conjugates.2. Binding experiments of CRM197-HRP to hCMEC/D3 cells showed that saturation of CRM197-HRP in hCMEC/D3 cells was reached after approximately 60 min.3. CRM197 can increase the permeability of BBB in a dose-dependent way and 300μg/kg CRM197 is the optimal dose to open the BBB in guinea pigs. With the increasing concentration of CRM197, the quantity of pinocytotic vesicles raised in brain microvascular endothelial cells in guinea pigs. After administration of CRM197 for 60 min, the DTR expressing level decreased in brain microvessel gradually.4. CRM197-HRP is delivered across the BBB by transcellular pathway. CRM197 mRNA reached a peak at 30 min and persisting for at least 60 min. Caveolin-1 protein levels were also up-regulated in response to CRM197.5. After the treatment of CRM197 for 60 min, the phosphorylation of Akt and FOXO1A was inhibited. The expression of p-Akt in cytoplasm and nucleus is attenuated and the distribution of FOXO1A in nucleus is increased.6. After the treatment of CRM197, TEER values were decreased gradually in BBB model in vitro. The immuno-staining of tight junction associated protein ZO-1 was attenuated in cellular boundaries and increased in cytoplasm in hCMEC/D3 cells. In brain microvessals, protein expressing level of ZO-1 and occludin decreased gradually. Claudin-5 protein expression reached the lowest level after the treatment of CRM197 for 30 min and then rised gradually.DiscussionOur results revealed that CRM197 not only delivered macromolecular substances HRP (40 kDa) across BBB in vitro, but also increased the permeability of BBB of guinea pig in a dose-dependent way. After the treatment of CRM197, the quantity of pinocytotic vesicles raised in brain microvascular endothelial cells. CRM197 increased the permeability of BBB by caveolae-mediated transcellular pathway, mRNA and protein expressing levels of CRM197 rised up in this process. CRM197 regulated caveolin-1 expressing levels through the inhibition of PI3K/Akt pathway and control of FOXO1A activity. CRM197 decreased the expression of tight junction associated proteins in brain microvessels, increased the redistribution of ZO-1 in cytoplasm and increased the permeability of BBB. In conclusion, CRM197 could deliver macromolecular substance across BBB through caveolae-mediated transcellular pathway and paracellular pathway.The sensitivity of mammalian cells to CRM197 varies among different mammalian species; humans, monkeys and guinea pigs are extremely sensitive whereas mouse and rat cells are resistant. In this research, hCMEC/D3 cells and guinea pigs were used as subjects, BBB models in vitro were established by hCMEC/D3 cells and were used to detect the association and internalization levels of CRM197-HRP in hCMEC/D3 cells. The intake of CRM197-HRP in hCMEC/D3 cells began from 5 min, and reached equilibrium in 60 min. Albumin or BSA are poorly transported across BBB and are often used as a marker to assess the permeability of BBB both in animal and in vitro studies. In this research, the binding of CRM197-HRP in hCMEC/D3 cells was approximately twofold higher than that of BSA-HRP, indicating a better endocytosis effect of CRM197-HRP. In the BBB model in vitro, CRM197-HRP was shown to prefer apical-to-basal transcytosis, although basal-to-apical could also occur. The apical-to-basal preference assured its potential utility in transporting CRM197 conjugates into brain tissue. CRM197 can increase the permeability of BBB in a dose-dependent way and there has no significant difference between 300μg/kg and 500μg/kg groups. To some potential safety considerations,300μg/kg CRM197 is the optimal dose to open the BBB in guinea pigs. After the treatment of CRM197, the quantity of pinocytotic vesicles raised in brain microvascular endothelial cells, which showed the transcellular pathway involved in CRM197 mediated transport.In BBB in vitro, transcytosis of CRM197-HRP could be inhibited by caveolae-mediated transcytosis inhibitor filipin, indicating that caveolae-mediated pathway was involved in the transport of the conjugates. CRM197 increased expression of caveolin-1 both in mRNA and in protein levels. The up-regulated caveolin-1 mRNA expression began at 5 min, and reached the peak at 30 min, accompanied by elevated caveolin-1 protein expressing levels. The induction of caveolin-1 mRNA and protein levels demonstrated an approximate two to threefold increase by CRM197. The up-regulated caveolin-1 expression may promote caveolae-mediated transcytosis and CRM197-targeted delivery across the BBB. As a scaffold protein, caveolin-1 interacts with many proteins such as growth factor receptors, signaling molecules, and regulates their function. Caveolin-1 expression is influenced by several signaling pathways. It has been shown that FOXO activation induces caveolin-1 expression through direct binding of FOXO to the caveolin-1 promoter region. PI3K/Akt pathway regulate FOXO activity. CRM197 could act as an HB-EGF inhibitor since it binds to DTR and blocks its proteolytic cleavage. Several signaling pathways induced by HB-EGF, like PI3K/Akt, can also be inhibited by CRM197. This results reveal that CRM197 could inhibit the PI3K/Akt pathway, reduce phospho-FOXO1A, and increase FOXO1A distribution in nuclei.Tight junction is an important structure of BBB to maintain the integrity and function. ZO-1, occludin and claudin-5 are the marker proteins of tight junction, and their expression and structure changes play a key role in the regulation of tight junction function. Our results showed the treatment of CRM197 attenuated the TEER value and the distributation of ZO-1 in cytomembrane, which demonstrated that CRM197 could regulate BBB permeability through paracellular pathway. After the treatment of CRM197 in vivo, ZO-1, occludin and claudin-5 expressing levels are down-regulated. In conclusion, CRM197 could increase the BBB permeability not only through caveolae-mediated transcellular pathway, but also through paracellular pathway.Our results showed that CRM197 could increase the permeability of BBB and mediated macromolecular substance transport across the BBB. Caveolae-mediated transcellular pathway and paracellular pathway involved in this process.Conclusions1. CRM197 could increase the permeability of BBB in a dose-dependent way and 300μg/kg CRM197 was the optimal dose to open the BBB. The quantity of pinocytotic vesicles raised in brain microvascular endothelial cells in this process.2. CRM197-HRP was delivered across the BBB in vitro effectively which was mediated by caveolae-dependent transcellular pathway.3. After the treatment of CRM197, the phosphorylation levels of Akt and FOXO1A were inhibited significantly and the distribution of FOXO1A in the nucleus was increased to up-regulate the expressing levels of caveolin-1.4. CRM197 could promote ZO-1 to redistribute in brain microvascular endothelial cells, significantly decreased the expressing levels of tight junction associated proteins ZO-1, occludin and claudin-5 to increase the permeability of BBB by paracellular pathway. |
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