Locally renewing resident synovial macrophages provide a protective barrier for the joint.

Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1- mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease.

The Basic Requirement of Tight Junction Proteins in Blood-Brain Barrier Function and Their Role in Pathologies.

This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.

Tight junction proteins at the blood-brain barrier: far more than claudin-5.

At the blood-brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.

Transmembrane proteins of the tight junctions at the blood-brain barrier: structural and functional aspects.

The blood-brain barrier (BBB) is formed by microvascular endothelial cells sealed by tetraspanning tight junction (TJ) proteins, such as claudins and TAMPs (TJ-associated marvel proteins, occludin and tricellulin). Claudins are the major components of the TJs. At the BBB, claudin-5 dominates the TJs by preventing the paracellular permeation of small molecules. On the other hand, TAMPs regulate the structure and function of the TJs; tricellulin may tighten the barrier for large molecules. This review aims at integrating and summarizing the most relevant and recent work on how the BBB is influenced by claudin-1, -3, -5, -12 and the TAMPs occludin and tricellulin, all of which are four-transmembrane TJ proteins. The exact functions of claudin-1, -3, -12 and TAMPs at this barrier still need to be elucidated.

Claudin-3 inhibits tumor-induced lymphangiogenesis via regulating the PI3K signaling pathway in lymphatic endothelial cells.

Claudin-3 is a tight junction protein that has often been associated with the progression and metastasis of various tumors. Here, the role of claudin-3 in tumor-induced lymphangiogenesis is investigated. We found an increased lymphangiogenesis in the B16F10 tumor in claudin-3 knockout mice, accompanied by augmented melanoma cell metastasis into sentinel lymph nodes. In vitro, the overexpression of claudin-3 on lymphatic endothelial cells inhibited tube formation by suppressing cell migration, resulting in restricted lymphangiogenesis. Further experiments showed that claudin-3 inhibited lymphatic endothelial cell migration by regulating the PI3K signaling pathway. Interestingly, the expression of claudin-3 in lymphatic endothelial cells is down-regulated by vascular endothelial growth factor C that is often present in the tumor microenvironment. This study indicates that claudin-3 plays an important role as a signaling molecule in lymphatic endothelial cell activity associated with tumor lymphangiogenesis, which may further contribute to melanoma metastasis.

Claudin-12 Deficiency Inhibits Tumor Growth by Impairing Transendothelial Migration of Myeloid-Derived Suppressor Cells.

Migration of myeloid-derived suppressor cells (MDSC) out of the circulation, across vascular walls, and into tumor is crucial for their immunosuppressive activity. A deeper understanding of critical junctional molecules and the regulatory mechanisms that mediate the extravasation of MDSCs could identify approaches to overcome cancer immunosuppression. In this study, we used mice deficient in tight junction protein Claudin-12 (Cldn12) compared with wild-type mice and found that loss of host Cldn12 inhibited the growth of transplanted tumors, reduced intratumoral accumulation of MDSCs, increased antitumor immune responses, and decreased tumor vascular density. Further studies revealed that Cldn12 expression on the cell surface of both MDSCs and endothelial cells (EC) is required for MDSCs transit across tumor vascular ECs. Importantly, expression of Cldn12 in MDSCs was modulated by GM-CSF in an AKT-dependent manner. Therefore, our results indicate that Cldn12 could serve as a promising target for restoring the antitumor response by interfering with MDSCs transendothelial migration. SIGNIFICANCE: Claudin-12-mediated homotypic interactions are critical for migration of myeloid-derived suppressor cells across vascular walls into tumor tissue, providing a potential therapeutic approach to overcome cancer immunosuppression.

Surrogate Cerebrospinal Fluid Biomarkers for Assessing the Efficacy of Gene Therapy in Hurler Syndrome.

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal hydroxylase alpha-l-iduronidase (IDUA). The resulting accumulation of dermatan and heparan sulfate induces intellectual disabilities and pre-mature death, and only a few treatment options are available. In a previous study, we demonstrated the feasibility, safety, and efficacy of gene therapy by injecting recombinant adeno-associated viral vector serotype (AAV)2/5-IDUA into the brain of a canine model of MPS I. We report on a quantitative proteomic analysis of control dogs and untreated dogs with MPS I cerebrospinal fluid (CSF) that had been collected throughout the study in the MPS I dogs. Mass spectrometry (MS) analysis identified numerous proteins present at altered levels in MPS I CSF samples. Quantitative immunoblotting, performed on CSF from healthy controls, untreated MPS I dogs, and MPS I dogs early treated and late treated by gene therapy, confirmed the MS data for a subset of proteins with higher abundance (neuronal pentraxin 1, chitinase 3-like 1, monocyte differentiation antigen CD14, and insulin-like growth factor-binding protein 2). Scoring of the results shows that the expression levels of these proteins are close to those of the control group for dogs that underwent gene therapy early in life but not for older treated animals. Our results disclose four novel predictive biomarker candidates that might be valuable in monitoring the course of the neurological disease in MPS patients at diagnosis, during clinical follow-up, and after treatment.

M01 as a novel drug enhancer for specifically targeting the blood-brain barrier.

Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.

Tight junctions in the blood-brain barrier promote edema formation and infarct size in stroke - Ambivalent effects of sealing proteins.

The outcome of stroke is greatly influenced by the state of the blood-brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.

Structure and function of claudins.

Claudins are tetraspan transmembrane proteins of tight junctions. They determine the barrier properties of this type of cell-cell contact existing between the plasma membranes of two neighbouring cells, such as occurring in endothelia or epithelia. Claudins can completely tighten the paracellular cleft for solutes, and they can form paracellular ion pores. It is assumed that the extracellular loops specify these claudin functions. It is hypothesised that the larger first extracellular loop is critical for determining the paracellular tightness and the selective ion permeability. The shorter second extracellular loop may cause narrowing of the paracellular cleft and have a holding function between the opposing cell membranes. Sequence analysis of claudins has led to differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and non-classic claudins (11-13, 16, 18, 20-24), according to their degree of sequence similarity. This is also reflected in the derived sequence-structure function relationships for extracellular loops 1 and 2. The concepts evolved from these findings and first tentative molecular models for homophilic interactions may explain the different functional contribution of the two extracellular loops at tight junctions.

A strategy for enrichment of claudins based on their affinity to Clostridium perfringens enterotoxin.

BACKGROUND: Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes. RESULTS: Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE(116-319) fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material. CONCLUSION: CPE(116-319) provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.

Quantitative Evaluation of Different Protein Fractions of Cerebrospinal Fluid Using 18O Labeling.

Molecular analysis of cerebrospinal fluid (CSF) provides comprehensive information on physiological and pathological processes related to the brain. In particular, proteomic studies give insights into the pathogenesis of many brain diseases which still pose diagnostic and therapeutic challenges. The identification of reliable biomarkers is an important step to meet these challenges. Mass spectrometry is an essential proteomic tool, not only for highly sensitive identification of proteins and posttranslational modifications, but also for their reliable quantification. Here, 18O labeling of tryptic peptides was employed to qualitative and quantitative analyses of protein fractions obtained by depletion of highly abundant proteins from cerebrospinal fluid. It was found that the execution of the investigated depletion protocols may cause the loss of potential protein biomarkers of neurological diseases.

Protective effects of peroxiredoxin-1 at the injured blood-brain barrier.

Reactive oxygen species (ROS) play a pivotal role in the development of neuroinflammatory disorders, such as multiple sclerosis (MS). Here, we studied the effect of ROS on protein expression in brain endothelial cells (BECs) using proteomic techniques and show that long-term exposure to ROS induces adaptive responses in BECs to counteract an oxidative attack. ROS induce differential protein expression in BECs, among which is peroxiredoxin-1 (Prx1). To further study the role of Prx1 we established a BEC line overexpressing Prx1. Our data indicate that Prx-1 overexpression protects BECs from ROS-induced cell death, reduces adhesion and subsequent transendothelial migration of monocytes by decreasing intercellular adhesion molecule-1 expression, and enhances the integrity of the BEC layer. Interestingly, vascular Prx1 immunoreactivity was markedly upregulated in inflammatory lesions of experimental autoimmune encephalomyelitis (EAE) animals and active demyelinating MS lesions. These findings indicate that enhanced vascular Prx1 expression may reflect the occurrence of vascular oxidative stress in EAE and MS. On the other hand, it may function as an endogenous defense mechanism to inhibit leukocyte infiltration and counteract ROS-induced cellular injury.

Cross-over endocytosis of claudins is mediated by interactions via their extracellular loops.

Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

Trictide, a tricellulin-derived peptide to overcome cellular barriers.

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a ß-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery.

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery.

The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 µmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between ß-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides.

The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account.

Depletion of highly abundant proteins from human cerebrospinal fluid: a cautionary note.

Affinity-based techniques, both for enrichment or depletion of proteins of interest, suffer from unwanted interactions between the bait or matrix material and molecules different from the original target. This effect was quantitatively studied by applying two common procedures for the depletion of albumin/gamma immunoglobulin to human cerebrospinal fluid. Proteins of the depleted and the column-bound fraction were identified by mass spectrometry, employing (18)O labeling for quantitation of their abundance. To different extents, the depletion procedures caused the loss of proteins previously suggested as biomarker candidates for neurological diseases. This is an important phenomenon to consider when quantifying protein levels in biological fluids.

Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia.

UNLABELLED: Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs; tricellulin, occludin, marvelD3) are thought to be relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. RESULTS: Ischemia, hypoxia, and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along the cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. INNOVATION: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts, as demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox dependently; a structural model is proposed. CONCLUSIONS: TAMP ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox sensor and sealing element at 3-cell contacts and may compensate as a redox mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox regulator for ischemia-related alterations.

Redox-sensitive structure and function of the first extracellular loop of the cell-cell contact protein claudin-1: lessons from molecular structure to animals.

UNLABELLED: The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. AIMS: We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. RESULTS: The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a ß-sheet binding surface flanked by an α-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. INNOVATION: The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding ß-sheet, stabilized by a shielded and redox-sensitive disulfide bond. CONCLUSION: Since the ß-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers.