白喉毒素无毒突变体CRM197介导大分子物质通过血脑屏障效果和机制的研究
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摘要
目的
血脑屏障的存在极大的限制了大分子药物通过血脑屏障进入脑组织,如何转运大分子药物通过血脑屏障是中枢神经系统疾病治疗亟待解决的一个关键问题。
目前研究表明,通过脑毛细血管内皮细胞上的内源性受体介导的药物转运可能是脑内药物转运最有效的方法之一。研究者们将药物或基因转移载体与一些内源性受体,如转铁蛋白或胰岛素受体的单克隆抗体偶联后,显示出高效的脑内转运效率。但由于上述单克隆抗体和体内的转铁蛋白、胰岛素等内源性配体能发生竞争性抑制作用,从而妨碍了脑组织摄取营养物质或药物。最新研究表明,白喉毒素的无毒突变体——交叉反应物质197 (cross-reacting material 197, CRM197)能够与分布在脑毛细血管内皮细胞上的白喉毒素受体(diphtheria toxin receptor, DTR)结合,转运大分子物质通过体外血脑屏障。由于CRM197作为荚膜多糖抗原载体蛋白或疫苗在国外已尝试应用于临床,而且在体内不存在与CRM197发生竞争性抑制作用的内源性配体,提示CRM197有可能成为新的更为有效的脑内药物转运载体。
药物通过血脑屏障一般通过两条途径：即跨细胞途径和细胞旁途径。在跨细胞途径中,质膜微囊蛋白caveolae依赖性跨细胞途径在药物通过血脑屏障的过程中起着重要的作用。Caveolin-1是caveolae的标志性蛋白,在维持caveolae的形态、结构和功能中起重要作用。研究表明,FOXO转录因子能够直接与caveolin-1的启动子区结合,调节caveolin-1的表达。FOXO转录因子的活性受磷脂酰肌醇3激酶(phospatidyl-3-inositol-kinase, PI3K)/丝氨酸／苏氨酸蛋白激酶(v-akt routine thymoma viral oncogene homolog, Akt)磷酸化级联通路的调节,激活的Akt使FOXO发生磷酸化修饰导致转录失活。CRM197与DTR结合后可以抑制受体剪切,显著减少肝素结合表皮生长因子(heparin-binding epidermal growth factor, HB-EGF)的形成,减弱由HB-EGF激活的PI3K/Akt等信号通路,进而可能参与对FOXO转录因子活性的调节,上调caveolin-1的表达。
药物通过血脑屏障的细胞旁途径主要是通过调节ZO-1 (zonula occluden-1)、occludin、claudin-5等紧密连接相关蛋白和粘附连接蛋白等结构,开放紧密连接,增加血脑屏障通透性。目前对于CRM197要通过何种途径介导大分子物质通过血脑屏障的研究尚未见报道。
本研究主要明确CRM197作用前后对体外和在体水平血脑屏障的通透性、跨细胞途径和细胞旁途径的影响,以及在此过程中PI3K/Akt/FOXO1A信号途径是否参与对caveolin-1表达的调节。
方法
1、体外血脑屏障模型和CRM197-HRP (horseradish peroxidase)、BSA-HRP偶联物的制备。
2、应用CRM197-HRP偶联物与人脑微血管内皮细胞hCMEC/D3的结合实验和跨细胞转运实验检测CRM197-HRP的转运效果。
3、应用Millicell-ERS电阻测量系统检测CRM197作用前后体外血脑屏障的跨内皮细胞电阻值transendothelial electric resistance, TEER)的变化。
4、应用伊文氏兰(Evans blue, EB)渗透性评估CRM197作用前后豚鼠血脑屏障通透性变化；透射电镜观察CRM197对脑微血管内皮细胞超微结构的影响。
5、应用免疫组织化学法和免疫荧光法检测CRM197作用前后p-Akt、p-FOXO1A和紧密连接相关蛋白ZO-1在hCMEC/D3细胞中的分布和表达水平的变化；以及caveolin-1和DTR在豚鼠脑微血管中的分布和表达水平的变化。
6、应用RT-PCR法检测CRM197作用前后hCMEC／D3细胞中caveolin-1 mRNA的表达水平；应用western blot法检测CRM197作用前后hCMEC/D3细胞和豚鼠脑微血管中caveolin-1、紧密连接相关蛋白ZO-1、occludin和claudin-5的蛋白表达水平以及PI3K/Akt/FOXO1A信号通路活性的变化。
结果
1、成功建立了体外血脑屏障模型,获得了CRM197-HRP偶联物。
2、CRM197-HRP与hCMEC/D3细胞的结合实验表明CRM197-HRP与hCMEC/D3细胞的结合水平呈时间依赖性增加,在60min时达到最大值。
3、CRM197能够以剂量依赖的方式增加豚鼠血脑屏障的通透性,其中300μg/kg CRM197为丌放血脑屏障的最适剂量。CRM197能够增加脑微血管内皮细胞中吞饮小泡的数量,以300μg/kg剂量组增加最明显。CRM197作用后,脑微血管中DTR的表达水平显著减少。
4、CRM197-HRP通过caveolae依赖性的跨细胞途径通过血脑屏障。Caveolin-1的mRNA和蛋白表达水平在CRM197作用后显著增加,在30min时达到峰值。
5、CRM197作用后,hCMEC/D3细胞中Akt和FOXO1A的蛋白磷酸化水平均显著下降,而总Akt的蛋白表达水平未发生变化；p-Akt在胞浆和胞核中的表达水平明显减弱；FOXO1A在细胞核中的分布增加。
6、CRM197作用60 min后,体外血脑屏障的TEER值显著下降,hCMEC/D3细胞中紧密连接相关蛋白ZO-1在细胞膜上的表达减弱,而在胞浆中的表达明显增强。CRM197作用60 min后,豚鼠脑微血管中ZO-1和occludin的蛋白表达水平均显著减少。Claudin-5的蛋白表达水平在CRM197作用30min时达到最低,而后增加。
讨论
本研究证明,CRM197不仅能介导大分子物质HRP (40 kDa)有效通过脑微血管内皮细胞,而且能以剂量依赖的方式增加豚鼠血脑屏障的通透性。CRM197作用后,脑微血管内皮细胞中吞饮小泡的数量增加。CRM197能够通过caveolae依赖的跨细胞途径增加血脑屏障通透性,这一过程伴有caveolin-1的mRNA和蛋白表达水平的增加,CRM197对PI3K/Akt信号途径的抑制以及对FOXO1A活性的调节能够参与对caveolin-1表达水平的调节。CRM197还能减少脑微血管中紧密连接相关蛋白ZO-1、occludin和claudin-5的表达,增加ZO-1在胞浆中的分布,增加血脑屏障通透性。综上所述,CRM197能够通过caveolae依赖性的跨细胞途径和细胞旁途径介导大分子物质通过血脑屏障。
由于不同种属动物的细胞对CRM197的敏感性不同,人、猴、豚鼠等对CRM197敏感,而大鼠和小鼠不敏感。所以本研究主要以hCMEC/D3细胞和豚鼠为研究对象。我们首先采用hCMEC/D3细胞建立了体外血脑屏障模型,并以此为研究对象检测了CRM197-HRP与hCMEC/D3细胞的特异性结合和内化水平,证实了hCMEC／D3细胞对CRM197-HRP的摄取在5min时开始增加,在60min左右达到平衡。由于血浆中的白蛋白分子量较大(66 kDa),很难通过血脑屏障,经常作为对照来评价体外或在体血脑屏障的通透性变化。本研究中,CRM197-HRP与hCMEC/D3细胞的结合水平约为BSA-HRP的两倍,表明CRM197-HRP具有更好的内吞效果。在体外血脑屏障模型中,CRM197-HRP与hCMEC/D3细胞结合后从transwell小室的上室到下室的转运水平要显著高于其从下室到上室的转运水平,这种转运方向的优势有助于转运CRM197偶联物进入脑组织。在体研究表明,CRM197能够以剂量依赖的方式增加豚鼠血脑屏障的通透性,其中300μg/kg和500μg／kg剂量组间EB含量未见显著差异。考虑到临床应用的安全性,我们采用300μg／kg剂量作为开放血脑屏障的最适剂量。CRM197作用后脑微血管中吞饮小泡的数量增加,提示跨细胞途径参与CRM197介导的转运过程。
通过对体外血脑屏障的研究证实,CRM197-HRP的跨细胞转运过程能够被caveolae依赖性胞吞转运抑制剂filipin所抑制,表明caveolae依赖性的跨细胞途径参与该偶联物的转运过程。CRM197作用后能增加caveolae标志蛋白caveolin-1的nRNA和蛋白表达水平。Caveolin-1的mRNA在CRM197作用5min时开始上调,在30min左右达到峰值,同时伴有caveolin-1蛋白表达水平的增加,其增加水平达到CRM197作用前的2-3倍左右。上调的caveolin-1能促进caveolae依赖性的跨细胞转运,有助于CRM197靶向转运大分子物质通过血脑屏障。Caveolin-1作为一个脚手架蛋白,能够直接与生长因子受体等多种蛋白和信号分子相互作用,参与对其功能的调节。Caveolin-1的表达也受到多种信号分子的影响,如FOXO转录因子能直接与caveolin-1的启动子区相结合,诱导caveolin-1的表达。FOXO转录因子的活性受PI3K/Akt磷酸化级联通路的调节。CRM197与白喉毒素受体结合后可以抑制受体剪切,显著减少剪切后HB-EGF的形成,减弱由HB-EGF激活的P13K／Akt等信号途径。本研究中,CRM197能通过抑制Akt的磷酸化水平,减少p-FOXO1A(Ser256)的表达,增加FOXO1A在细胞核中的分布而上调caveolin-1的表达。
紧密连接是维持体内血脑屏障完整性重要的结构和功能基础。ZO-1、occludin和claudin-5是紧密连接的标志蛋白,这些蛋白的表达、结构或功能的变化对紧密连接起着重要的调节作用。我们在体外研究中发现,CRM197作用后能引起内皮细胞的TEER值改变,同时减少ZO-1在细胞膜上的分布,表明CRM197也能通过细胞旁途径调节血脑屏障的通透性。通过在体实验进一步证实CRM197作用后伴有紧密连接相关蛋白ZO-1、occludin和claudin-5表达水平的下调。以上研究表明,CRM197不仅通过caveolae依赖性的跨细胞途径,而且通过细胞旁途径增加血脑屏障的通透性。
上述研究结果证明,CRM197能增加血脑屏障的通透性,介导大分子物质转运通过血脑屏障。这一过程既有caveolae依赖性跨细胞途径也有细胞旁途径的参与。
结论
1、CRM197能够以剂量依赖的方式增加血脑屏障通透性,300μg/kg CRM197为开放血脑屏障的最适剂量,这一过程伴有脑微血管内皮细胞中吞饮小泡数量的增加。
2、CRM197-HRP能有效通过体外血脑屏障,这一过程主要与caveolae依赖性的跨细胞途径有关。
3、CRM197能够显著抑制脑微血管内皮细胞中Akt和FOXO1A的磷酸化水平,增加FOXO1A在细胞核中的分布,上调caveolin-1的表达。
4、CRM197能够调节紧密连接相关蛋白ZO-1在脑微血管内皮细胞中的分布,显著减少紧密连接相关蛋白ZO-1、occludin和claudin-5的表达,通过细胞旁途径增加血脑屏障通透性。
Objective
Blood-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.
Methods
1. 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.
Results
1. 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.
Discussion
Our 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.
Conclusions
1. 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.
血脑屏障的存在极大的限制了大分子药物通过血脑屏障进入脑组织,如何转运大分子药物通过血脑屏障是中枢神经系统疾病治疗亟待解决的一个关键问题。
目前研究表明,通过脑毛细血管内皮细胞上的内源性受体介导的药物转运可能是脑内药物转运最有效的方法之一。研究者们将药物或基因转移载体与一些内源性受体,如转铁蛋白或胰岛素受体的单克隆抗体偶联后,显示出高效的脑内转运效率。但由于上述单克隆抗体和体内的转铁蛋白、胰岛素等内源性配体能发生竞争性抑制作用,从而妨碍了脑组织摄取营养物质或药物。最新研究表明,白喉毒素的无毒突变体——交叉反应物质197 (cross-reacting material 197, CRM197)能够与分布在脑毛细血管内皮细胞上的白喉毒素受体(diphtheria toxin receptor, DTR)结合,转运大分子物质通过体外血脑屏障。由于CRM197作为荚膜多糖抗原载体蛋白或疫苗在国外已尝试应用于临床,而且在体内不存在与CRM197发生竞争性抑制作用的内源性配体,提示CRM197有可能成为新的更为有效的脑内药物转运载体。
药物通过血脑屏障一般通过两条途径：即跨细胞途径和细胞旁途径。在跨细胞途径中,质膜微囊蛋白caveolae依赖性跨细胞途径在药物通过血脑屏障的过程中起着重要的作用。Caveolin-1是caveolae的标志性蛋白,在维持caveolae的形态、结构和功能中起重要作用。研究表明,FOXO转录因子能够直接与caveolin-1的启动子区结合,调节caveolin-1的表达。FOXO转录因子的活性受磷脂酰肌醇3激酶(phospatidyl-3-inositol-kinase, PI3K)/丝氨酸／苏氨酸蛋白激酶(v-akt routine thymoma viral oncogene homolog, Akt)磷酸化级联通路的调节,激活的Akt使FOXO发生磷酸化修饰导致转录失活。CRM197与DTR结合后可以抑制受体剪切,显著减少肝素结合表皮生长因子(heparin-binding epidermal growth factor, HB-EGF)的形成,减弱由HB-EGF激活的PI3K/Akt等信号通路,进而可能参与对FOXO转录因子活性的调节,上调caveolin-1的表达。
药物通过血脑屏障的细胞旁途径主要是通过调节ZO-1 (zonula occluden-1)、occludin、claudin-5等紧密连接相关蛋白和粘附连接蛋白等结构,开放紧密连接,增加血脑屏障通透性。目前对于CRM197要通过何种途径介导大分子物质通过血脑屏障的研究尚未见报道。
本研究主要明确CRM197作用前后对体外和在体水平血脑屏障的通透性、跨细胞途径和细胞旁途径的影响,以及在此过程中PI3K/Akt/FOXO1A信号途径是否参与对caveolin-1表达的调节。
方法
1、体外血脑屏障模型和CRM197-HRP (horseradish peroxidase)、BSA-HRP偶联物的制备。
2、应用CRM197-HRP偶联物与人脑微血管内皮细胞hCMEC/D3的结合实验和跨细胞转运实验检测CRM197-HRP的转运效果。
3、应用Millicell-ERS电阻测量系统检测CRM197作用前后体外血脑屏障的跨内皮细胞电阻值transendothelial electric resistance, TEER)的变化。
4、应用伊文氏兰(Evans blue, EB)渗透性评估CRM197作用前后豚鼠血脑屏障通透性变化；透射电镜观察CRM197对脑微血管内皮细胞超微结构的影响。
5、应用免疫组织化学法和免疫荧光法检测CRM197作用前后p-Akt、p-FOXO1A和紧密连接相关蛋白ZO-1在hCMEC/D3细胞中的分布和表达水平的变化；以及caveolin-1和DTR在豚鼠脑微血管中的分布和表达水平的变化。
6、应用RT-PCR法检测CRM197作用前后hCMEC／D3细胞中caveolin-1 mRNA的表达水平；应用western blot法检测CRM197作用前后hCMEC/D3细胞和豚鼠脑微血管中caveolin-1、紧密连接相关蛋白ZO-1、occludin和claudin-5的蛋白表达水平以及PI3K/Akt/FOXO1A信号通路活性的变化。
结果
1、成功建立了体外血脑屏障模型,获得了CRM197-HRP偶联物。
2、CRM197-HRP与hCMEC/D3细胞的结合实验表明CRM197-HRP与hCMEC/D3细胞的结合水平呈时间依赖性增加,在60min时达到最大值。
3、CRM197能够以剂量依赖的方式增加豚鼠血脑屏障的通透性,其中300μg/kg CRM197为丌放血脑屏障的最适剂量。CRM197能够增加脑微血管内皮细胞中吞饮小泡的数量,以300μg/kg剂量组增加最明显。CRM197作用后,脑微血管中DTR的表达水平显著减少。
4、CRM197-HRP通过caveolae依赖性的跨细胞途径通过血脑屏障。Caveolin-1的mRNA和蛋白表达水平在CRM197作用后显著增加,在30min时达到峰值。
5、CRM197作用后,hCMEC/D3细胞中Akt和FOXO1A的蛋白磷酸化水平均显著下降,而总Akt的蛋白表达水平未发生变化；p-Akt在胞浆和胞核中的表达水平明显减弱；FOXO1A在细胞核中的分布增加。
6、CRM197作用60 min后,体外血脑屏障的TEER值显著下降,hCMEC/D3细胞中紧密连接相关蛋白ZO-1在细胞膜上的表达减弱,而在胞浆中的表达明显增强。CRM197作用60 min后,豚鼠脑微血管中ZO-1和occludin的蛋白表达水平均显著减少。Claudin-5的蛋白表达水平在CRM197作用30min时达到最低,而后增加。
讨论
本研究证明,CRM197不仅能介导大分子物质HRP (40 kDa)有效通过脑微血管内皮细胞,而且能以剂量依赖的方式增加豚鼠血脑屏障的通透性。CRM197作用后,脑微血管内皮细胞中吞饮小泡的数量增加。CRM197能够通过caveolae依赖的跨细胞途径增加血脑屏障通透性,这一过程伴有caveolin-1的mRNA和蛋白表达水平的增加,CRM197对PI3K/Akt信号途径的抑制以及对FOXO1A活性的调节能够参与对caveolin-1表达水平的调节。CRM197还能减少脑微血管中紧密连接相关蛋白ZO-1、occludin和claudin-5的表达,增加ZO-1在胞浆中的分布,增加血脑屏障通透性。综上所述,CRM197能够通过caveolae依赖性的跨细胞途径和细胞旁途径介导大分子物质通过血脑屏障。
由于不同种属动物的细胞对CRM197的敏感性不同,人、猴、豚鼠等对CRM197敏感,而大鼠和小鼠不敏感。所以本研究主要以hCMEC/D3细胞和豚鼠为研究对象。我们首先采用hCMEC/D3细胞建立了体外血脑屏障模型,并以此为研究对象检测了CRM197-HRP与hCMEC/D3细胞的特异性结合和内化水平,证实了hCMEC／D3细胞对CRM197-HRP的摄取在5min时开始增加,在60min左右达到平衡。由于血浆中的白蛋白分子量较大(66 kDa),很难通过血脑屏障,经常作为对照来评价体外或在体血脑屏障的通透性变化。本研究中,CRM197-HRP与hCMEC/D3细胞的结合水平约为BSA-HRP的两倍,表明CRM197-HRP具有更好的内吞效果。在体外血脑屏障模型中,CRM197-HRP与hCMEC/D3细胞结合后从transwell小室的上室到下室的转运水平要显著高于其从下室到上室的转运水平,这种转运方向的优势有助于转运CRM197偶联物进入脑组织。在体研究表明,CRM197能够以剂量依赖的方式增加豚鼠血脑屏障的通透性,其中300μg/kg和500μg／kg剂量组间EB含量未见显著差异。考虑到临床应用的安全性,我们采用300μg／kg剂量作为开放血脑屏障的最适剂量。CRM197作用后脑微血管中吞饮小泡的数量增加,提示跨细胞途径参与CRM197介导的转运过程。
通过对体外血脑屏障的研究证实,CRM197-HRP的跨细胞转运过程能够被caveolae依赖性胞吞转运抑制剂filipin所抑制,表明caveolae依赖性的跨细胞途径参与该偶联物的转运过程。CRM197作用后能增加caveolae标志蛋白caveolin-1的nRNA和蛋白表达水平。Caveolin-1的mRNA在CRM197作用5min时开始上调,在30min左右达到峰值,同时伴有caveolin-1蛋白表达水平的增加,其增加水平达到CRM197作用前的2-3倍左右。上调的caveolin-1能促进caveolae依赖性的跨细胞转运,有助于CRM197靶向转运大分子物质通过血脑屏障。Caveolin-1作为一个脚手架蛋白,能够直接与生长因子受体等多种蛋白和信号分子相互作用,参与对其功能的调节。Caveolin-1的表达也受到多种信号分子的影响,如FOXO转录因子能直接与caveolin-1的启动子区相结合,诱导caveolin-1的表达。FOXO转录因子的活性受PI3K/Akt磷酸化级联通路的调节。CRM197与白喉毒素受体结合后可以抑制受体剪切,显著减少剪切后HB-EGF的形成,减弱由HB-EGF激活的P13K／Akt等信号途径。本研究中,CRM197能通过抑制Akt的磷酸化水平,减少p-FOXO1A(Ser256)的表达,增加FOXO1A在细胞核中的分布而上调caveolin-1的表达。
紧密连接是维持体内血脑屏障完整性重要的结构和功能基础。ZO-1、occludin和claudin-5是紧密连接的标志蛋白,这些蛋白的表达、结构或功能的变化对紧密连接起着重要的调节作用。我们在体外研究中发现,CRM197作用后能引起内皮细胞的TEER值改变,同时减少ZO-1在细胞膜上的分布,表明CRM197也能通过细胞旁途径调节血脑屏障的通透性。通过在体实验进一步证实CRM197作用后伴有紧密连接相关蛋白ZO-1、occludin和claudin-5表达水平的下调。以上研究表明,CRM197不仅通过caveolae依赖性的跨细胞途径,而且通过细胞旁途径增加血脑屏障的通透性。
上述研究结果证明,CRM197能增加血脑屏障的通透性,介导大分子物质转运通过血脑屏障。这一过程既有caveolae依赖性跨细胞途径也有细胞旁途径的参与。
结论
1、CRM197能够以剂量依赖的方式增加血脑屏障通透性,300μg/kg CRM197为开放血脑屏障的最适剂量,这一过程伴有脑微血管内皮细胞中吞饮小泡数量的增加。
2、CRM197-HRP能有效通过体外血脑屏障,这一过程主要与caveolae依赖性的跨细胞途径有关。
3、CRM197能够显著抑制脑微血管内皮细胞中Akt和FOXO1A的磷酸化水平,增加FOXO1A在细胞核中的分布,上调caveolin-1的表达。
4、CRM197能够调节紧密连接相关蛋白ZO-1在脑微血管内皮细胞中的分布,显著减少紧密连接相关蛋白ZO-1、occludin和claudin-5的表达,通过细胞旁途径增加血脑屏障通透性。
Objective
Blood-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.
Methods
1. 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.
Results
1. 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.
Discussion
Our 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.
Conclusions
1. 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.
引文
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10 Shinefield HR, Black S, Ray P, et al. Safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate vaccine in infants and toddlers. Pediatr Infect Dis J.1999; 18(9):757-763.
11 Frank PG, Pavlides S, Lisanti MP. Caveolae and transcytosis in endothelial cells:role in atherosclerosis. Cell Tissue Res.2009;335(1):41-47.
12 Doherty GJ, McMahon HT. Mechanisms of endocytosis. Annu Rev Biochem.2009; 78:857-902.
13 Xia CY, Zhang Z, Xue YX, et al. Mechanisms of the increase in the permeability of the blood-tumor barrier obtained by combining low-frequency ultrasound irradiation with small-dose bradykinin. J Neurooncol.2009;94(1):41-50
14 Ikezu T, Ueda H, Trapp BD, et al. Affinity-purification and characterization of caveolins from the brain:differential expression of caveolin-1,-2, and-3 in brain endothelial and astroglial cell types. Brain Res.1998;804:177-192.
15 Virgintino D, Robertson D, Errede M, et al. Expression of caveolin-1 in human brain microvessels. Neuroscience.2002;115:145-152.
16 Wu D, Terrian DM. Regulation of caveolin-1 expression and secretion by a protein kinase cepsilon signaling pathway in human prostate cancer cells. J Biol Chem.2002; 277:40449-40455.
17 Xie Z, Zeng X, Waldman T, et al. Transformation of mammary epithelial cells by 3-phosphoinositide-dependent protein kinase-1 activates beta-catenin and c-Myc, and down-regulates caveolin-1. Cancer Res.2003; 63:5370-5375.
18 van den Heuvel AP, Schulze A, Burgering BM. Direct control of caveolin-1 expression by FOXO transcription factors. J Biochem.2005; 385:795-802.
19 Zanella F, Link W, Carnero A. Understanding FOXO, New Views on Old Transcription Factors. Curr Cancer Drug Targets.2010 Jan 1. [Epub ahead of print]
20 Abbott NJ, Patabendige AA, Dolman DE, et al. Structure and function of the blood-brain barrier. Neurobiol Dis.2010; 37(1):13-25.
21 Dorland RB, Middlebrook JL, Leppla SH. Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells. J Biol chem.1979; 254(22):11337-11342.
22 Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci.2006;7(1):41-53.
23 Deli MA, Abraham CS, Kataoka Y, et al. Permeability studies on in vitro blood-brain barrier models:physiology, pathology, and pharmacology. Cell Mol Neurobiol.2005;25(1):59-127.
24 Candela P, Gosselet F, Miller F, et al. Physiological pathway for low-density lipoproteins across the blood-brain barrier:transcytosis through brain capillary endothelial cells in vitro. Endothelium.2008; 15(5-6):254-264.
25 Dehouck B, Fenart L, Dehouck MP, et al. A new function for the LDL receptor:transcytosis of LDL across the blood-brain barrier. J Cell Biol.1997; 138(4):877-889.
26 Ikezu T, Ueda H, Trapp BD, et al. Affinity-purification and characterization of caveolins from the brain:differential expression of caveolin-1,-2, and-3 in brain endothelial and astroglial cell types. Brain Res.1998;804:177-192.
27 Virgintino D, Robertson D, Errede M, et al. Expression of caveolin-1 in human brain microvessels. Neuroscience.2002;115:145-152.
28 Razani B, Lisanti MP. Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A. Am J Physiol Cell Physiol.2001; 281:C1241-1250.
29 Li S, Couet J, Lisanti MP. Src tyrosine kinases, Ga subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J Biol Chem.1996; 271:29182-29190.
30 Roy UK, Henkhaus RS, Ignatenko NA, et al. Wild-type APC regulates caveolin-1 expression in human colon adenocarcinoma cell lines via FOXO1a and C-myc. Mol Carcinog.2008; 47(12):947-955.
31 Lee HY, You HJ, Won JY, et al. Forkhead factor, FOXO3a, induces apoptosis of endothelial cells through activation of matrix metalloproteinases. Arterioscler Thromb Vasc Biol.2008; 28:302-308.
32 Zhao YY, Gao XP, Zhao YD, et al. Endothelial cell-restricted disruption of FoxMl impairs endothelial repair following LPS-induced vascular injury. J Clin Invest.2006; 116:2333-2343.
33 Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell.1999; 96:857-868.
34 Tang ED, Nunez G, Barr FG, et al. Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem.1999; 274:16741-16746.
35 Nag S, Venugopalan R, Stewart DJ. Increased caveolin-1 expression precedes decreased expression of occludin and claudin-5 during blood-brain barrier breakdown. Acta Neuropathol. 2007;114(5):459-469.
36 Zhong Y, Smart EJ, Weksler B, et al. Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling. J Neurosci.2008;28(31):7788-7796.
37 Boado RJ, Zhang YF, Zhang Y, et al. Humanization of anti-human insulin receptor antibody for drug targeting across the human blood-brain barrier. Biotechnol Bioeng.2007; 96(2):381-391.
38 Pardridge WM. Drug targeting to the brain. Pharm Res.2007; 24(9):1733-1744.
39 Cha JH, Brooke JS, Eidels L. Hamster diphtheria toxin receptor:a naturally occurring chimera of monkey and mouse HB-EGF precursors. Biochem Biophys Res Commun.1999; 254(2):325-329.
40 Cha JH, Chang MY, Richardson JA, et al. Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin. Mol Microbiol.2003;49(1):235-240.
41 Cohen KA, Liu TF, Cline JM, et al. Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey. Cancer Immunol Immunother. 2005;54(8):799-806.
42 Couet J, Li S, Okamoto T, et al. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae associated proteins. J Biol Chem.1997;272:6525-6633.
43 Gloor SM, Wachtel M, Bolliger MF, et al. Molecular and cellular permeability control at the blood-brain barrier. Brain Res Brain Res Rev.2001;36(2-3):258-264.
44 Wolburg H, Lippoldt A. Tight junctions of the blood-brain barrier:development, composition and regulation. Vascul Pharmacol.2002;38(6):323-337.
45 Hartsock A, Nelson WJ. Adherens and tight junctions:structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta.2008;1778(3):660-669.
46 Bauer H, Zweimueller-Mayer J, Steinbacher P, et al. The dual role of zonula occludens (ZO) proteins. J Biomed Biotechnol.2010; 2010:402593. Epub 2010 Mar 9.
47 Furuse M. Molecular basis of the core structure of tight junctions. Cold Spring Harb Perspect Biol.2010; 2(1):a002907.
1 Engelhardt B, Sorokin L. The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction. Semin Immunopathol.2009; 31(4):497-511.
2 Banks WA. Blood-brain barrier as a regulatory interface. Forum Nutr.2010; 63:102-110.
3 Blakeley J. Drug delivery to brain tumors. Curr Neurol Neurosci Rep.2008; 8(3):235-241.
4 De Boer AG, Gaillard PJ. Strategies to improve drug delivery across the blood-brain barrier. Clin Pharmacokinet.2007;46(7):553-576.
5 Cornford EM, Hyman S. Blood-brain barrier permeability to small and large molecules. Adv Drug Del Rev.1999;36(2-3):145-163.
6 Pavan B, Dalpiaz A, Ciliberti N, et al. Progress in drug delivery to the central nervous system by the prodrug approach. Molecules.2008;13(5):1035-1065.
7 Lorke DE, Kalasz H, Petroianu GA, et al. Entry of oximes into the brain:a review. Curr Med Chem.2008;15(8):743-53.
8 Ardridge WM. Receptor-mediated peptide transport through the blood-brain barrier. Endocr Rev.1986;7(4):314-430.
9 Baines AC, Zhang B. Receptor-mediated protein transport in the early secretory pathway. Trends Biochem Sci.2007;32(8):381-388.
10 Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov.2002;1(2):131-139.
11 Banks WA. Enhanced leptin transport across the blood-brain barrier by alpha1-adrenergic agents. Brain Res.2001;899(1-2):209-217.
12 Moos T, Rosengren Nielsen T, Skjorringe T, et al. Iron trafficking inside the brain. J Neurochem.2007;103(5):1730-1740.
13 Tamai I, T sujiA. Drug delivery through the blood-brain barrier. Adv Drug Del Rev.1996; 19(3):401-402.
14 Deguchi Y, Naito T, Yuge T, et al. Blood-brain barrier transport of 1251-labeled basic fibroblast growth factor. Pharm Res.2000; 17:63-69.
15 Lu W, Wan J, She Z, et al. Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle. J Control Release.2007; 118(1):38-53.
16 Haverstick K, Fleming A, Mark Saltzman W. Conjugation to increase treatment volume during local therapy:a case study with PEGylated camptothecin. Bioconjug Chem.2007; 18(6):2115-2121.
17 Schmidt PG, Campbell KM, Hinds KD, et al. PEGylated bioactive molecules in biodegradable polymer microparticles. Expert Opin Biol Ther.2007; 7(9):1427-1436.
18 Veronese FM, Mero A. The impact of PEGylation on biological therapies. BioDrugs.2008; 22(5):315-329.
19 Hamidi M, Azadi A, Rafiei P. Pharmacokinetic consequences of pegylation. Drug Deliv. 2006;13(6):399-409.
20 Parveen S, Sahoo SK. Nanomedicine:clinical applications of polyethylene glycol conjugated proteins and drugs. Clin Pharmacokinet.2006; 45(10):965-988.
21 Parveen S, Sahoo SK. Nanomedicine:clinical applications of polyethylene glycol conjugated proteins and drugs. Clin Pharmacokinet.2006;45(10):965-988.
22 Greenwald RB, Choe YH, McGuire J, et al. Effective drug delivery by PEGylated drug conjugates. Adv Drug Deliv Rev.2003;55(2):217-250.
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24 Aktas Y, Yemisci M,Andrieux K, et al. Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26. Bioconjug Chem.2005; 16(6):1503-1511.
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35 Gaillarda PJ, Brinka A, De Boer AG. Diphtheria toxin receptor-targeted brain drug delivery. Int Cong Ser.2005;1277:185-198.
36 Gaillard PJ, de Boer AG. A novel opportunity for targeted drug delivery to the brain. J Control Release.2006; 116(2):e60-e62.
37 Choe S, Bennett M, Fujii G, et al. The crystal structure of diphtheria toxin. Nature.1992, 357:216-222.
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44 Fogar P, Navaglia F, Basso D, et al. Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197:implications for pancreatic cancer gene therapy. Cancer Gene Ther.2010;17(1):58-68.
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48 Martarelli D, Pompei P, Mazzoni G. Inhibition of adrenocortical carcinoma by diphtheria toxin mutant CRM 197. Chemotherapy.2009; 55(6):425-432.
49 Shimura T, Kataoka H, Ogasawara N, et al. Diphtheria toxin mutant CRM 197 possesses weak EF2-ADP-ribosyl activity that potentiates its anti-tumorigenic activity. J Biochem. 2007; 142(1):95-104.
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57 Mitamura T, Higashiyama S, Taniguchi N, et al. Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/ diphtheria toxin receptor and inhibits specifically its mitogenic activity. J Biol Chem.1995; 270(3):1015-1019.
58 Raab G, Klagsbrun M. Heparin-binding EGF-likegrowth factor. Biochim Biophys Acta. 1997; 1333(3):F179-199.
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60 Tanaka N, Sasahara M, Ohno M, et al. Heparinbinding epidermal growth factor-like growth factor mRNA expression in neonatal rat brain with hypoxic/ischemic injury. Brain Res.1999;827(1-2):130-138.
2 Gabathuler R. Approaches to transport therapeutic drugs across the blood-brain barrier to treat brain diseases. Neurobiol Dis.2010; 37(1):48-57.
3 Jones AR, Shusta EV. Blood-brain barrier transport of therapeutics via receptor-mediation. Pharm Res.2007;24(9):1759-1771.
4 Gaillarda PJ, Brinka A, De Boer AG. Diphtheria toxin receptor-targeted brain drug delivery. Int Cong Ser.2005;1277:185-198.
5 Chansel D, Ciroldi M, Vandermeersch S, et al. Heparin binding EGF is necessary for vasospastic response to endothelin. FASEB J.2006; 20(11):1936-1938.
6 Iwamoto R, Mekada E. Heparin-binding EGF-like growth factor:a juxtacrine growth factor. Cytokine Growth Factor Rev.2000; 11(4):335-344.
7 Miyamoto S, Hirata M, Yamazaki A, et al. Heparin-binding EGF-like growth factor is a promising target for ovarian cancer therapy. Cancer Res.2004; 64(16):5720-5727.
8 Martarelli D, Pompei P, Mazzoni G. Inhibition of adrenocortical carcinoma by diphtheria toxin mutant CRM197. Chemotherapy.2009; 55(6):425-432.
9 Dagan R, Poolman JT, Zepp F. Combination vaccines containing DTPa-Hib:impact of IPV and coadministration of CRM197 conjugates. Expert Rev Vaccines.2008; 7(1):97-115.
10 Shinefield HR, Black S, Ray P, et al. Safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate vaccine in infants and toddlers. Pediatr Infect Dis J.1999; 18(9):757-763.
11 Frank PG, Pavlides S, Lisanti MP. Caveolae and transcytosis in endothelial cells:role in atherosclerosis. Cell Tissue Res.2009;335(1):41-47.
12 Doherty GJ, McMahon HT. Mechanisms of endocytosis. Annu Rev Biochem.2009; 78:857-902.
13 Xia CY, Zhang Z, Xue YX, et al. Mechanisms of the increase in the permeability of the blood-tumor barrier obtained by combining low-frequency ultrasound irradiation with small-dose bradykinin. J Neurooncol.2009;94(1):41-50
14 Ikezu T, Ueda H, Trapp BD, et al. Affinity-purification and characterization of caveolins from the brain:differential expression of caveolin-1,-2, and-3 in brain endothelial and astroglial cell types. Brain Res.1998;804:177-192.
15 Virgintino D, Robertson D, Errede M, et al. Expression of caveolin-1 in human brain microvessels. Neuroscience.2002;115:145-152.
16 Wu D, Terrian DM. Regulation of caveolin-1 expression and secretion by a protein kinase cepsilon signaling pathway in human prostate cancer cells. J Biol Chem.2002; 277:40449-40455.
17 Xie Z, Zeng X, Waldman T, et al. Transformation of mammary epithelial cells by 3-phosphoinositide-dependent protein kinase-1 activates beta-catenin and c-Myc, and down-regulates caveolin-1. Cancer Res.2003; 63:5370-5375.
18 van den Heuvel AP, Schulze A, Burgering BM. Direct control of caveolin-1 expression by FOXO transcription factors. J Biochem.2005; 385:795-802.
19 Zanella F, Link W, Carnero A. Understanding FOXO, New Views on Old Transcription Factors. Curr Cancer Drug Targets.2010 Jan 1. [Epub ahead of print]
20 Abbott NJ, Patabendige AA, Dolman DE, et al. Structure and function of the blood-brain barrier. Neurobiol Dis.2010; 37(1):13-25.
21 Dorland RB, Middlebrook JL, Leppla SH. Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells. J Biol chem.1979; 254(22):11337-11342.
22 Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci.2006;7(1):41-53.
23 Deli MA, Abraham CS, Kataoka Y, et al. Permeability studies on in vitro blood-brain barrier models:physiology, pathology, and pharmacology. Cell Mol Neurobiol.2005;25(1):59-127.
24 Candela P, Gosselet F, Miller F, et al. Physiological pathway for low-density lipoproteins across the blood-brain barrier:transcytosis through brain capillary endothelial cells in vitro. Endothelium.2008; 15(5-6):254-264.
25 Dehouck B, Fenart L, Dehouck MP, et al. A new function for the LDL receptor:transcytosis of LDL across the blood-brain barrier. J Cell Biol.1997; 138(4):877-889.
26 Ikezu T, Ueda H, Trapp BD, et al. Affinity-purification and characterization of caveolins from the brain:differential expression of caveolin-1,-2, and-3 in brain endothelial and astroglial cell types. Brain Res.1998;804:177-192.
27 Virgintino D, Robertson D, Errede M, et al. Expression of caveolin-1 in human brain microvessels. Neuroscience.2002;115:145-152.
28 Razani B, Lisanti MP. Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A. Am J Physiol Cell Physiol.2001; 281:C1241-1250.
29 Li S, Couet J, Lisanti MP. Src tyrosine kinases, Ga subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J Biol Chem.1996; 271:29182-29190.
30 Roy UK, Henkhaus RS, Ignatenko NA, et al. Wild-type APC regulates caveolin-1 expression in human colon adenocarcinoma cell lines via FOXO1a and C-myc. Mol Carcinog.2008; 47(12):947-955.
31 Lee HY, You HJ, Won JY, et al. Forkhead factor, FOXO3a, induces apoptosis of endothelial cells through activation of matrix metalloproteinases. Arterioscler Thromb Vasc Biol.2008; 28:302-308.
32 Zhao YY, Gao XP, Zhao YD, et al. Endothelial cell-restricted disruption of FoxMl impairs endothelial repair following LPS-induced vascular injury. J Clin Invest.2006; 116:2333-2343.
33 Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell.1999; 96:857-868.
34 Tang ED, Nunez G, Barr FG, et al. Negative regulation of the forkhead transcription factor FKHR by Akt. J Biol Chem.1999; 274:16741-16746.
35 Nag S, Venugopalan R, Stewart DJ. Increased caveolin-1 expression precedes decreased expression of occludin and claudin-5 during blood-brain barrier breakdown. Acta Neuropathol. 2007;114(5):459-469.
36 Zhong Y, Smart EJ, Weksler B, et al. Caveolin-1 regulates human immunodeficiency virus-1 Tat-induced alterations of tight junction protein expression via modulation of the Ras signaling. J Neurosci.2008;28(31):7788-7796.
37 Boado RJ, Zhang YF, Zhang Y, et al. Humanization of anti-human insulin receptor antibody for drug targeting across the human blood-brain barrier. Biotechnol Bioeng.2007; 96(2):381-391.
38 Pardridge WM. Drug targeting to the brain. Pharm Res.2007; 24(9):1733-1744.
39 Cha JH, Brooke JS, Eidels L. Hamster diphtheria toxin receptor:a naturally occurring chimera of monkey and mouse HB-EGF precursors. Biochem Biophys Res Commun.1999; 254(2):325-329.
40 Cha JH, Chang MY, Richardson JA, et al. Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin. Mol Microbiol.2003;49(1):235-240.
41 Cohen KA, Liu TF, Cline JM, et al. Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey. Cancer Immunol Immunother. 2005;54(8):799-806.
42 Couet J, Li S, Okamoto T, et al. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae associated proteins. J Biol Chem.1997;272:6525-6633.
43 Gloor SM, Wachtel M, Bolliger MF, et al. Molecular and cellular permeability control at the blood-brain barrier. Brain Res Brain Res Rev.2001;36(2-3):258-264.
44 Wolburg H, Lippoldt A. Tight junctions of the blood-brain barrier:development, composition and regulation. Vascul Pharmacol.2002;38(6):323-337.
45 Hartsock A, Nelson WJ. Adherens and tight junctions:structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta.2008;1778(3):660-669.
46 Bauer H, Zweimueller-Mayer J, Steinbacher P, et al. The dual role of zonula occludens (ZO) proteins. J Biomed Biotechnol.2010; 2010:402593. Epub 2010 Mar 9.
47 Furuse M. Molecular basis of the core structure of tight junctions. Cold Spring Harb Perspect Biol.2010; 2(1):a002907.
1 Engelhardt B, Sorokin L. The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction. Semin Immunopathol.2009; 31(4):497-511.
2 Banks WA. Blood-brain barrier as a regulatory interface. Forum Nutr.2010; 63:102-110.
3 Blakeley J. Drug delivery to brain tumors. Curr Neurol Neurosci Rep.2008; 8(3):235-241.
4 De Boer AG, Gaillard PJ. Strategies to improve drug delivery across the blood-brain barrier. Clin Pharmacokinet.2007;46(7):553-576.
5 Cornford EM, Hyman S. Blood-brain barrier permeability to small and large molecules. Adv Drug Del Rev.1999;36(2-3):145-163.
6 Pavan B, Dalpiaz A, Ciliberti N, et al. Progress in drug delivery to the central nervous system by the prodrug approach. Molecules.2008;13(5):1035-1065.
7 Lorke DE, Kalasz H, Petroianu GA, et al. Entry of oximes into the brain:a review. Curr Med Chem.2008;15(8):743-53.
8 Ardridge WM. Receptor-mediated peptide transport through the blood-brain barrier. Endocr Rev.1986;7(4):314-430.
9 Baines AC, Zhang B. Receptor-mediated protein transport in the early secretory pathway. Trends Biochem Sci.2007;32(8):381-388.
10 Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov.2002;1(2):131-139.
11 Banks WA. Enhanced leptin transport across the blood-brain barrier by alpha1-adrenergic agents. Brain Res.2001;899(1-2):209-217.
12 Moos T, Rosengren Nielsen T, Skjorringe T, et al. Iron trafficking inside the brain. J Neurochem.2007;103(5):1730-1740.
13 Tamai I, T sujiA. Drug delivery through the blood-brain barrier. Adv Drug Del Rev.1996; 19(3):401-402.
14 Deguchi Y, Naito T, Yuge T, et al. Blood-brain barrier transport of 1251-labeled basic fibroblast growth factor. Pharm Res.2000; 17:63-69.
15 Lu W, Wan J, She Z, et al. Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle. J Control Release.2007; 118(1):38-53.
16 Haverstick K, Fleming A, Mark Saltzman W. Conjugation to increase treatment volume during local therapy:a case study with PEGylated camptothecin. Bioconjug Chem.2007; 18(6):2115-2121.
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