Matrix stiffness regulates the tight junction phenotypes and local barrier properties in tricellular regions in an iPSC-derived BBB model.

The blood-brain barrier (BBB) can respond to various mechanical cues such as shear stress and substrate stiffness. In the human brain, the compromised barrier function of the BBB is closely associated with a series of neurological disorders that are often also accompanied by the alteration of brain stiffness. In many types of peripheral vasculature, higher matrix stiffness decreases barrier function of endothelial cells through mechanotransduction pathways that alter cell-cell junction integrity. However, human brain endothelial cells are specialized endothelial cells that largely resist changes in cell morphology and key BBB markers. Therefore, it has remained an open question how matrix stiffness affects barrier integrity in the human BBB. To gain insight into the effects of matrix stiffness on BBB permeability, we differentiated brain microvascular endothelial-like cells from human induced pluripotent stem cells (iBMEC-like cells) and cultured the cells on extracellular matrix-coated hydrogels of varying stiffness. We first detected and quantified the junction presentation of key tight junction (TJ) proteins. Our results show matrix-dependent junction phenotypes in iBMEC-like cells, where cells on softer gels (1 kPa) have significantly lower continuous and total TJ coverages. We also determined that these softer gels also lead to decreased barrier function in a local permeability assay. Furthermore, we found that matrix stiffness regulates the local permeability of iBMEC-like cells through the balance of continuous ZO-1 TJs and no junction regions ZO-1 in tricellular regions. Together, these findings provide valuable insights into the effects of matrix stiffness on TJ phenotypes and local permeability of iBMEC-like cells. STATEMENT OF SIGNIFICANCE: Brain mechanical properties, including stiffness, are particularly sensitive indicators for pathophysiological changes in neural tissue. The compromised function of the blood-brain barrier is closely associated with a series of neurological disorders often accompanied by altered brain stiffness. In this study, we use polymeric biomaterials and provide new evidence that biomaterial stiffness regulates the local permeability in iPSC-derived brain endothelial cells in tricellular regions through the tight junction protein ZO-1. Our findings provide valuable insights into the changes in junction architecture and barrier permeability in response to different substrate stiffnesses. Since BBB dysfunction has been linked to many diseases, understanding the influence of substrate stiffness on junction presentations and barrier permeability could lead to the development of new treatments for diseases associated with BBB dysfunction or drug delivery across BBB systems.

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality-aspects of infection that may contribute to pathogenesis in vivo.

Photodynamic Priming Modulates Endothelial Cell-Cell Junction Phenotype for Light-activated Remote Control of Drug Delivery.

The blood-brain barrier (BBB) remains a major obstacle for drug delivery to the central nervous system. In particular, the tight and adherens junctions that join the brain capillary endothelial cells limit the diffusion of various molecules from the bloodstream into the brain. Photodynamic priming (PDP) is a non-cytotoxic modality that involves light activation of photosensitizers to photochemically modulate nearby molecules without killing the cells. Here we investigate the effects of sub-lethal photochemistry on junction phenotype (i.e., continuous, punctate, or perpendicular), as well as the BBB permeability in a transwell model of human brain microvascular endothelial cells (HBMECs). We showed that PDP decreases the continuous junction architecture by ~20%, increases the perpendicular junction architecture by ~40%, and has minimal impact on cell morphology in HBMECs. Furthermore, transwell permeability assay revealed that PDP improves the HBMEC permeability to dextran or nanoliposomes by up to 30-fold for 6-9 days. These results suggest that PDP could safely reverse the mature brain endothelial junctions without killing the HBMECs. This study not only emphasizes the critical roles of PDP in the modulation junction phenotype, but also highlights the opportunity to further develop PDP-based combinations that opens the cerebrum endothelium for enhanced drug transporter across the BBB.

Quantitatively relating brain endothelial cell-cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program.

BACKGROUND: The endothelial cell-cell junctions of the blood-brain barrier (BBB) play a pivotal role in the barrier's function. Altered cell-cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell-cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a "global" and "local" scale. METHODS: We cultured HBMECs on collagen I, fibronectin, collagen IV, laminin, fibronectin/collagen IV/laminin, or hyaluronic acid/gelatin for 2, 4, and 7 days with varying cAMP treatment schedules. Images of immunostained ZO-1, VE-cadherin, and claudin-5 were analyzed using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. Transwell permeability assays and resistance measurements were used to measure bulk ("global") barrier properties, and a "local" permeability assay was used to correlate junction presentation proximal to permeable monolayer regions. RESULTS: Substrate composition was found to play little role in junction presentation, while cAMP supplements significantly increased the continuous junction architecture. Increased culture time required increased cAMP treatment time to reach similar ZO-1 and VE-cadherin coverage observed with shorter culture, though longer cultures were required for claudin-5 presentation. Prolonged cAMP treatment (6 days) disrupted junction integrity for all three junction proteins. Transwell permeability and TEER assays showed no correlation with junction phenotype, but a local permeability assay revealed a correlation between the number of discontinuous and no junction regions with barrier penetration. CONCLUSIONS: These results suggest that cAMP signaling influences HBMEC junction architecture more than matrix composition. Our studies emphasized the need for local barrier measurement to mechanistically understand the role of junction phenotype and supported previous results that continuous junctions are indicative of a more mature/stable endothelial barrier. Understanding what conditions influence junction presentations, and how they, in turn, affect barrier integrity, could lead to the development of therapeutics for diseases associated with BBB dysfunction.

Tumor Cell Mechanosensing During Incorporation into the Brain Microvascular Endothelium.

INTRODUCTION: Tumor metastasis to the brain occurs in approximately 20% of all cancer cases and often occurs due to tumor cells crossing the blood-brain barrier (BBB). The brain microenvironment is comprised of a soft hyaluronic acid (HA)-rich extracellular matrix with an elastic modulus of 0.1-1 kPa, whose crosslinking is often altered in disease states. METHODS: To explore the effects of HA crosslinking on breast tumor cell migration, we developed a biomimetic model of the human brain endothelium, consisting of brain microvascular endothelial cell (HBMEC) monolayers on HA and gelatin (HA/gelatin) films with different degrees of crosslinking, as established by varying the concentration of the crosslinker Extralink. RESULTS AND DISCUSSION: Metastatic breast tumor cell migration speed, diffusion coefficient, spreading area, and aspect ratio increased with decreasing HA crosslinking, a mechanosensing trend that correlated with tumor cell actin organization but not CD44 expression. Meanwhile, breast tumor cell incorporation into endothelial monolayers was independent of HA crosslinking density, suggesting that alterations in HA crosslinking density affect tumor cells only after they exit the vasculature. Tumor cells appeared to exploit both the paracellular and transcellular routes of trans-endothelial migration. Quantitative phenotyping of HBMEC junctions via a novel Python software revealed a VEGF-dependent decrease in punctate VE-cadherin junctions and an increase in continuous and perpendicular junctions when HBMECs were treated with tumor cell-secreted factors. CONCLUSIONS: Overall, our quantitative results suggest that a combination of biochemical and physical factors promote tumor cell migration through the BBB.

Praseodymium-142 microspheres for brachytherapy of nonresectable hepatic tumors.

PURPOSE: To perform dosimetric study of (142)Pr microspheres for the use as a possible choice of radionuclide in microsphere brachytherapy of nonresectable hepatic tumor for faster dose delivery and facilitated dosimetry for quality assurance. METHODS AND MATERIALS: Dose distributions of (142)Pr and (90)Y microspheres within hepatic tumors and blood vessels were calculated using MCNPX2.6 Monte Carlo code. The biological effective doses (BEDs) for (142)Pr and (90)Y microspheres were calculated and compared using the linear-quadratic model. RESULTS: Dose distributions due to beta particles were similar for both (142)Pr and (90)Y. Total initial activity required to achieve the same total dose of 150 Gy at 2 cm from the center of the tumor was 0.662 GBq and 0.191 GBq for (142)Pr and (90)Y, respectively. For α/ß ratio equal to 10 Gy, calculated BED values were 301.0 and 194.7 for (142)Pr and (90)Y, respectively, considering a total physical dose of 150 Gy. CONCLUSIONS: Total dose delivery and dose distributions for both (142)Pr and (90)Y within tumors and blood vessels were obtained and compared. Shorter half-life of (142)Pr is an advantage, enabling a faster dose delivery. The higher BED found for (142)Pr implies potential improvement in the treatment effectiveness. (142)Pr showed to be an attractive option for applications in microsphere brachytherapy.