Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 µm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 µm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

Human Vascular Wall Microfluidic Model for Preclinical Evaluation of Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical animal models often leads to candidate compound termination during drug development. DIVI has not been documented in human clinical trials with drugs that cause DIVI in preclinical animals. A robust human preclinical assay for DIVI is needed as an early vascular injury screen. A human vascular wall microfluidic tissue chip was developed with a human umbilical vein endothelial cell (HUVEC)-umbilical artery smooth muscle cell (vascular smooth muscle cell, VSMC) bilayer matured under physiological shear stress. Optimized temporal flow profiles produced HUVEC-VSMC bilayers with quiescent endothelial cell (EC) monolayers, EC tight junctions, and contractile VSMC morphology. Dose-response testing (3-30 µM concentration) was conducted with minoxidil and tadalafil vasodilators. Both drugs have demonstrated preclinical DIVI but lack clinical evidence. The permeability of severely damaged engineered bilayers (30 µM tadalafil) was 4.1 times that of the untreated controls. Immunohistochemical protein assays revealed contrasting perspectives on tadalafil and minoxidil-induced damage. Tadalafil impacted the endothelial monolayer with minor injury to the contractile VSMCs, whereas minoxidil demonstrated minor EC barrier injury but damaged VSMCs and activated ECs in a dose-response manner. This proof-of-concept human vascular wall bilayer model of DIVI is a critical step toward developing a preclinical human screening assay for drug development. Impact statement More than 90% of drug candidates fail during clinical trials due to human efficacy and toxicity concerns. Preclinical studies rely heavily on animal models, although animal toxicity and drug metabolism responses often differ from humans. During the drug development process, perfused in vitro human tissue chips could model the clinical drug response and potential toxicity of candidate compounds. Our long-term objective is to develop a human vascular wall tissue chip to screen for drug-induced vascular injury. Its application could ultimately reduce drug development delays and costs, and improve patient safety.

Effect of Pore Size of Honeycomb-Patterned Polymer Film on Spontaneous Formation of 2D Micronetworks by Coculture of Human Umbilical Vein Endothelial Cells and Mesenchymal Stem Cells.

The geometrical control of micronetwork structures ( µ NSs) formed by endothelial cells is an important topic in tissue engineering, cell-based assays, and fundamental biological studies. In this study, µ NSs are formed using human umbilical vein endothelial cells (HUVECs) by the coculture of HUVECs and human mesenchymal stem cells (MSCs) confined in a honeycomb-patterned poly-l-lactic acid film (honeycomb film (HCF)), which is a novel cell culture scaffold. The HCF is produced using the breath figure method, which uses condensed water droplets as pore templates. The confinement of the HUVECs and MSCs in the HCF along with the application of centrifugal force results in µ NS formation when the pore size is more than 20  µ m. Furthermore, µ NS development is geometrically restricted by the hexagonally packed and connected pores in the horizontal direction of the HCF. Network density is also controlled by changing the seeding density of the HUVECs and MSCs. The threshold pore size indicates that µ NSs can be formed spontaneously by using an HCF with a perfectly uniform porous structure. This result provides an important design guideline for the structure of porous cell culture scaffolds by applying a blood vessel model in vitro.

Time-of-day-dependent variations of scratching behavior and transepidermal water loss in mice that developed atopic dermatitis.

Scratching and skin barrier dysfunctions are pivotal features and therapeutic targets of atopic dermatitis (AD); however, time-of-day-dependent variations of these characteristics remain unclear. NC/Tnd mice have been shown to exhibit severe scratching behavior and skin barrier disruption together with the development of spontaneous atopic dermatitis when they are raised under air-uncontrolled environment. In the present study, time-of-day-dependent variations of scratching behavior and transepidermal water loss (TEWL) were evaluated in NC/Tnd mice that developed moderate to severe AD. Analysis of the mice for 24 hr revealed that scratching frequency and duration were increased from in the afternoon to the nocturnal period when locomotor activity was low, and scratching behavior was decreased in the morning. The highest scratching frequency and duration were 3.8- and 4.1-fold increases in the lowest scratching frequency and duration, respectively. In addition, TEWL on the dorsal skin lesion was decreased in the diurnal period, while that was increased in the nocturnal period. The highest TEWL was a 1.3-fold increase in the lowest TEWL. Significant daily variations were detected in scratching frequency and duration and TEWL. These results indicate that NC/Tnd mice are an appropriate mouse model to investigate time-of-day-dependent variations of scratching behavior and skin barrier dysfunctions associated with AD.

Circadian rhythms and the effect of glucocorticoids on expression of the clock gene period1 in canine peripheral blood mononuclear cells.

Circadian rhythms have a periodicity of approximately 24h and, in mammals, are regulated by clock genes. In this study, expression profiles of clock genes (per1, per2, clock, bmal1 and cry1) were investigated over a single 24h period by real-time PCR in peripheral blood mononuclear cells (PBMCs) of healthy dogs and canine PBMCs treated in vitro and in vivo with glucocorticoids. Only per1 mRNA exhibited daily rhythms in canine PBMCs. Canine PBMCs cultured with dexamethasone in vitro had dose- and time-dependent increases in per1 mRNA expression. Intravenous injection of dexamethasone increased expression of per1 in canine PBMCs in vivo. Rhythmic expression of per1 in PBMCs could be used as a molecular marker for monitoring circadian rhythms and the effects of drugs on clock genes in dogs.