Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides

Abstract

The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.

Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers

“…In concordance with the functional measurements of barrier opening, cytokine treatment caused a decreased immunostaining at cellular borders, where punctate and discontinuous staining was observed (Fig 4). In our previous work we also demonstrated morphological changes at cellular junctions in parallel with opening of the paracellular cleft in Caco-2 cells by TJ modulator peptides [12]. In agreement with our observations claudin-4 was described to promote barrier function in different cell cultures, and proinflammatory cytokines decreased its junctional presence and increased the proportion of mobile claudin-4 in the TJ of Caco-2 cells [33,34].…”

“…Treatment of Caco-2 cells with TNF-α or IL-1β decrease the electrical resistance of monolayers and increase IL-8 production indicating epithelial barrier opening and inflammatory response [8,10,11]. In our previous study we described, that claudin-4, a sealing claudin, is the most expressed member of the claudin family after claudin-1 in Caco-2 cells [12]. Claudin-4 was described as an important element of the intestinal barrier in both colon tissue of mice and Caco-2 cells with a significant downregulation in inflammation [13].…”

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery

“…The observations presented here suggest that internalization of C5C2 followed by downregulation and redistribution of junctional proteins results in TJ and barrier opening. The alterations in claudin‐5 cytochemistry, as well as in the endothelial morphology dominated by claudin‐5, clearly indicate disturbances of claudin interactions. Earlier studies demonstrate that claudin‐5 peptides are mainly internalized by caveolae‐mediated endocytosis and to a minor extent by micropinocytosis and clathrin‐mediated endocytosis .…”

“…Claudin‐5, a main component of the TJs in the brain endothelium, plays a major role in paracellular tightening of the BBB . In this study, we used peptide segments of the ECL1 of claudin‐5 (C5C2 and derivatives) as templates to design peptidomimetics for modulating the parent protein (i.e., for transient and reversible opening of the BBB to enhance drug delivery).…”

“…Next, 8 × 10 4 endothelial cells/cm 2 were seeded on the upside of the membranes and plated to 12‐well plates containing glial cells. The three cell types were cultured together for 4–5 days …”