ANGPTL7 and Its Role in IOP and Glaucoma.

Purpose: Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in IOP homeostasis and its potential as a target for glaucoma therapeutics. Methods: IOP, outflow facility, and outflow tissue morphology of Angptl7 knockout (KO) mice were assessed with and without dexamethasone (Dex). ANGPTL7 was quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids. Antibodies to ANGPTL7 were generated and tested in three-dimensional (3D) culture of outflow cells and perfused human donor eyes. Rabbits were injected intravitreally with a neutralizing antibody targeting ANGPTL7, and IOP was measured. Results: IOP was significantly elevated, but outflow facility and outflow tissue morphology were not different between Angptl7 KO mice and littermates. When challenged with Dex, IOP increased in wild-type but not Angptl7 KO mice. In human samples, increased ANGPTL7 was seen in the aqueous humor of patients treated with steroids, regardless of glaucoma status. Using 3D culture, recombinant ANGPTL7 decreased, and ANGPTL7-blocking antibodies increased hydraulic conductivity. Significantly, outflow facility increased in human eyes treated ex vivo with ANGPTL7-blocking antibodies, and IOP decreased for 21 days in rabbits after a single injection of blocking antibodies. Conclusions: Using multiple models, we have demonstrated that excess ANGPTL7 increases outflow resistance and IOP and that neutralizing ANGPTL7 has beneficial effects in both naïve and steroid-induced hypertensive eyes, thus motivating the development of ANGPTL7-targeting therapeutics for the treatment of glaucoma.

Engineering cryoelectrospun elastin-alginate scaffolds to serve as stromal extracellular matrices.

Scaffold-based regenerative strategies that emulate physical, biochemical, and mechanical properties of the native extracellular matrix (ECM) of the region of interest can influence cell growth and function. Existing ECM-mimicking scaffolds, including nanofiber (NF) mats, sponges, hydrogels, and NF-hydrogel composites are unable to simultaneously mimic typical composition, topography, pore size, porosity, and viscoelastic properties of healthy soft-tissue ECM. In this work, we used cryoelectrospinning to fabricate 3D porous scaffolds with minimal fibrous backbone, pore size and mechanical properties similar to soft-tissue connective tissue ECM. We used salivary glands as our soft tissue model and found the decellularized adult salivary gland (DSG) matrix to have a fibrous backbone, 10-30µm pores, 120 Pa indentation modulus, and ∼200 s relaxation half time. We used elastin and alginate as natural, compliant biomaterials and water as the solvent for cryoelectrospinning scaffolds to mimic the structure and viscoelasticity of the connective tissue ECM of the DSG. Process parameters were optimized to produce scaffolds with desirable topography and compliance similar to DSG, with a high yield of >100 scaffolds/run. Using water as solvent, rather than organic solvents, was critical to generate biocompatible scaffolds with desirable topography; further, it permitted a green chemistry fabrication process. Here, we demonstrate that cryoelectrospun scaffolds (CESs) support penetration of NIH 3T3 fibroblasts 250-450µm into the scaffold, cell survival, and maintenance of a stromal cell phenotype. Thus, we demonstrate that elastin-alginate CESs mimic many structural and functional properties of ECM and have potential for future use in regenerative medicine applications.

Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation.

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial-mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial-mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell-cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.