Epilepsy is the third most common chronic neurological disorder. Clinical and experimental evidence supports the role of sex and influence of sex hormones on seizures and epilepsy as well as alterations of the endocrine system and levels of sex hormones by epileptiform activity. Conversely, seizures are sensitive to changes in sex hormone levels, which in turn may affect the seizure-induced neuronal damage. The effects of reproductive hormones on neuronal excitability and seizure-induced damage are complex to contradictory and depend on different mechanisms, which have to be accounted for in data interpretation. Both estradiol and progesterone/allopregnanolone may have beneficial effects for patients with epilepsy. Individualized hormonal therapy should be considered as adjunctive treatment in patients with epilepsy to improve seizure control as well as quality of life.

Perfusion-independent regulation of epithelial pattern formation by the vasculature during organ development and regeneration is of considerable interest for application in restoring organ function. During murine submandibular salivary gland development, the vasculature co-develops with the epithelium during branching morphogenesis; however, it is not known whether the vasculature has instructive effects on the epithelium. Using pharmacological inhibitors and siRNA knockdown in embryonic organ explants, we determined that VEGFR2-dependent signaling is required for salivary gland epithelial patterning. To test directly for a requirement for endothelial cells in instructive epithelial patterning, we developed a novel ex vivo cell fractionation/reconstitution assay. Immunodepletion of CD31 + endothelial cells in this assay confirmed a requirement for endothelial cells in epithelial patterning of the gland. Depletion of endothelial cells or inhibition of VEGFR2 signaling in organ explants caused an aberrant increase in cells expressing the ductal proteins K19 and K7, with a reduction in Kit + progenitor cells in the endbuds of reconstituted glands. Addition of exogenous endothelial cells to reconstituted glands restored epithelial patterning, as did supplementation with the endothelial cell-regulated mesenchymal factors IGFBP2 and IGFBP3. Our results demonstrate that endothelial cells promote expansion of Kit + progenitor cells and suppress premature ductal differentiation in early developing embryonic submandibular salivary gland buds.

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10µL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.

Mechanical properties of the microenvironment regulate cell morphology and differentiation within complex organs. However, methods to restore morphogenesis and differentiation in organs in which compliance is suboptimal are poorly understood. We used mechanosensitive mouse salivary gland organ explants grown at different compliance levels together with deoxycholate extraction and immunocytochemistry of the intact, assembled matrices to examine the compliance-dependent assembly and distribution of the extracellular matrix and basement membrane in explants grown at permissive or non-permissive compliance. Extracellular matrix and basement membrane assembly were disrupted in the glands grown at low compliance compared to those grown at high compliance, correlating with defective morphogenesis and decreased myoepithelial cell differentiation. Extracellular matrix and basement membrane assembly as well as myoepithelial differentiation were restored by addition of TGFβ1 and by mechanical rescue, and mechanical rescue was prevented by inhibition of TGFβ signaling during the rescue. We detected a basal accumulation of active integrin β1 in the differentiating myoepithelial cells that formed a continuous peripheral localization around the proacini and in clefts within active sites of morphogenesis in explants that were grown at high compliance. The pattern and levels of integrin β1 activation together with myoepithelial differentiation were interrupted in explants grown at low compliance but were restored upon mechanical rescue or with application of exogenous TGFβ1. These data suggest that therapeutic application of TGFβ1 to tissues disrupted by mechanical signaling should be examined as a method to promote organ remodeling and regeneration.