A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions

Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.

Effects of Ruscus extract on muscarinic receptors: Is there a role for endothelium derived relaxing factors on macromolecular permeability protection and microvascular diameter changes?

BACKGROUND: Protective effects of Ruscus extract on macromolecular permeability depend on its capacity to stimulate muscarinic receptors on endothelial cells and induce the release of endothelium derived relaxing factors (EDRFs). OBJECTIVE: To investigate if these effects depend only on activation of muscarinic receptors or EDRFs release are also necessary. We have also investigated the participation of Ruscus extract on muscarinic-induced release of EDRFs on microvascular diameters. METHODS: Hamsters were treated daily during two weeks with Ruscus extract (50, 150 and 450 mg/kg/day) and then macromolecular permeability induced by histamine and arteriolar and venular diameters after cyclooxygenase (COX) and nitric oxide synthase (NOS) inhibitors: indomethacin and Nω-Nitro-L-arginine (LNA), respectively applied topically at 10–8M, 10–6M and 10–4M were observed on the cheek pouch preparation. RESULTS: Ruscus extract decreased macromolecular permeability in a dose-dependent fashion and did not affect microvascular diameters. NOS and COX inhibitors enhanced this effect. NOS inhibition reduced arteriolar diameter and COX blocking decreased arteriolar and venular diameters at the lowest dose and increased them at higher doses of Ruscus extract. CONCLUSION: The protective effect of Ruscus extract on macromolecular permeability seems to be mediated only via muscarinic receptors. Muscarinic activation attenuated vasoconstrictive tone through cyclooxygenase-independent endothelium derived relaxing factors.

From Farm to Pharmacy: Strawberry-Enabled Oral Delivery of Protein Drugs

Although oral drug delivery is preferred by patients, it is not possible for proteins because the gastrointestinal tract is not sufficiently permeable. To enable the non-toxic oral uptake of protein drugs, we investigated plant-based foods as intestinal permeation enhancers, hypothesizing that compounds found in food would be well-tolerated by the gastrointestinal tract. Following a screen of over 100 fruits, vegetables, herbs, and fungi, we identified strawberry as a potent enhancer of macromolecular permeability in vitro and in mice. Natural product chemistry techniques identified pelargonidin, an anthocyanidin, as the active compound. In mice, insulin was orally administered with pelargonidin to induce sustained pharmacodynamic effects with doses as low as 1 U/kg and bioactivity of over 100% relative to the current gold standard of subcutaneous injection. Pelargonidin-induced permeability was reversible within two hours of treatment, and one month of daily dosing did not adversely affect mice as determined by weight tracking, serum concentrations of inflammatory markers, and tight junction gene expression. Results underscore the utility of plant-based foods in biomedical applications and demonstrate pelargonidin as an especially potent enhancer for the oral delivery of biologics.

In the absence of overt urothelial damage, chondroitinase ABC digestion of the GAG layer increases bladder permeability in ovariectomized female rats

Loss of integrity of the protective impermeability barrier in the urothelium has been identified as significant in bladder dysfunction. In this study, we tested the theory that the luminal layer of glycosaminoglycans (GAG) serves as an important component of barrier function. The peptide polycation protamine sulfate (PS), 1 mg/ml, was instilled intravesically for 10 min into rat bladders. Chondroitinase ABC (ChABC), 63 IU/ml, was instilled into an additional six rats for 30 min to digest the GAG layer. Unmanipulated controls and sham-injected controls were also performed. After 24 h, the rats were euthanized, the bladders were removed, and permeability was assessed in the Ussing chamber and by diffusion of FITC-labeled dextran (4 kDa) to measure macromolecular permeability. The status of tight junctions was assessed by immunofluorescence and electron microscopy. In control and sham treated rat bladders, the transepithelial electrical resistance were means of 2.5 ± 1.1 vs. 2.6 ± 1.1 vs 1.2 ± 0.5 and 1.01 ± 0.7 kΩ·cm2 in the PS-treated and ChABC-treated rat bladders ( P = 0.0016 and P = 0.0039, respectively). Similar differences were seen in dextran permeability. Histopathology showed a mild inflammation following PS treatment, but the ChABC-treated bladders were indistinguishable from controls. Tight junctions generally remained intact. ChABC digestion alone induced bladder permeability, confirming the importance of the GAG layer to bladder barrier function and supports that loss of the GAG layer seen in bladder biopsies of interstitial cystitis patients could be a significant factor producing symptoms for at least some interstitial cystitis/painful bladder syndrome patients.

Oxytocin regulates gastrointestinal motility, inflammation, macromolecular permeability, and mucosal maintenance in mice

Enteric neurons express oxytocin (OT); moreover, enteric neurons and enterocytes express developmentally regulated OT receptors (OTRs). Although OT (with secretin) opposes intestinal inflammation, physiological roles played by enteric OT/OTR signaling have not previously been determined. We tested hypotheses that OT/OTR signaling contributes to enteric nervous system (ENS)-related gastrointestinal (GI) physiology. GI functions and OT effects were compared in OTR-knockout (OTRKO) and wild-type (WT) mice. Stool mass and water content were greater in OTRKO mice than in WT. GI transit time in OTRKO animals was faster than in WT; OT inhibited in vitro generation of ENS-dependent colonic migrating motor complexes in WT but not in OTRKO mice. Myenteric neurons were hyperplastic in OTRKO animals, and mucosal exposure to cholera toxin (CTX) in vitro activated Fos in more myenteric neurons in OTRKO than WT than in WT mice; OT inhibited the CTX response in WT but not in OTRKO mice. Villi and crypts were shorter in OTRKO than in WT mice, and transit-amplifying cell proliferation in OTRKO crypts was deficient. Macromolecular intestinal permeability in OTRKO was greater than WT mice, and experimental colitis was more severe in OTRKO mice; moreover, OT protected WT animals from colitis. Observations suggest that OT/OTR signaling acts as a brake on intestinal motility, decreases mucosal activation of enteric neurons, and promotes enteric neuronal development and/or survival. It also regulates proliferation of crypt cells and mucosal permeability; moreover OT/OTR signaling is protective against inflammation. Oxytocinergic signaling thus appears to play an important role in multiple GI functions that are subject to neuronal regulation.