FGF Gradient Controls Boundary Position Between Proliferating and Differentiating Cells and Regulates Lacrimal Gland Growth Dynamics

Fibroblast growth factor (FGF) signaling plays an important role in controlling cell proliferation, survival, and cell movements during branching morphogenesis of many organs. In mammals branching morphogenesis is primarily regulated by members of the FGF7-subfamily (FGF7 and FGF10), which are expressed in the mesenchyme, and signal to the epithelial cells through the “b” isoform of fibroblast growth factor receptor-2 (FGFR2). Our previous work demonstrated that FGF7 and FGF10 form different gradients in the extracellular matrix (ECM) and induce distinct cellular responses and gene expression profiles in the lacrimal and submandibular glands. The last finding was the most surprising since both FGF7 and FGF10 bind signal most strongly through the same fibroblast growth factor receptor-2b isoform (FGFR2b). Here we revisit this question to gain an explanation of how the different FGFs regulate gene expression. For this purpose, we employed our ex vivo epithelial explant migration assay in which isolated epithelial explants are grown near the FGF loaded beads. We demonstrate that the graded distribution of FGF induces activation of ERK1/2 MAP kinases that define the position of the boundary between proliferating “bud” and differentiating “stalk” cells of growing lacrimal gland epithelium. Moreover, we showed that gene expression profiles of the epithelial explants exposed to distinct FGFs strictly depend on the ratio between “bud” and “stalk” area. Our data also suggests that differentiation of “stalk” and “bud” regions within the epithelial explants is necessary for directional and persistent epithelial migration. Gaining a better understanding of FGF functions is important for development of new approaches to enhance tissue regeneration

Lacrimal Gland Repair Using Progenitor Cells

Abstract In humans, the lacrimal gland (LG) is the primary contributor to the aqueous layer of the tear film. Production of tears in insufficient quantity or of inadequate quality may lead to aqueous‐deficiency dry eye (ADDE). Currently there is no cure for ADDE. The development of strategies to reliably isolate LG stem/progenitor cells from the LG tissue brings great promise for the design of cell replacement therapies for patients with ADDE. We analyzed the therapeutic potential of epithelial progenitor cells (EPCPs) isolated from adult wild‐type mouse LGs by transplanting them into the LGs of TSP ‐1−/− mice, which represent a novel mouse model for ADDE. TSP‐1−/− mice are normal at birth but progressively develop a chronic form of ocular surface disease, characterized by deterioration, inflammation, and secretory dysfunction of the lacrimal gland. Our study shows that, among c‐kit‐positive epithelial cell adhesion molecule (EpCAM+) populations sorted from mouse LGs, the c‐kit+dim/EpCAM+/Sca1−/CD34−/CD45− cells have the hallmarks of an epithelial cell progenitor population. Isolated EPCPs express pluripotency factors and markers of the epithelial cell lineage Runx1 and EpCAM, and they form acini and ducts when grown in reaggregated three‐dimensional cultures. Moreover, when transplanted into injured or “diseased” LGs, they engraft into acinar and ductal compartments. EPCP‐injected TSP‐1−/− LGs showed reduction of cell infiltration, differentiation of the donor EPCPs within secretory acini, and substantial improvement in LG structural integrity and function. This study provides the first evidence for the effective use of adult EPCP cell transplantation to rescue LG dysfunction in a model system. Stem Cells Translational Medicine 2017;6:88–9

Role of Matrix Metalloproteinases 2 and 9 in Lacrimal Gland Disease in Animal Models of Sjögren's Syndrome

Citation: Aluri HS, Kublin CL, Thotakura S, et al. Role of matrix metalloproteinases 2 and 9 in lacrimal gland disease in animal models of Sjögren's syndrome. Invest Ophthalmol Vis Sci. 2015;56:5218-5228. DOI:10.1167/ iovs.15-17003 PURPOSE. Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. METHODS. The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. RESULTS. There was a 2.5-and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. CONCLUSIONS. We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands

Double-staining of β-taxilin and desmin proteins.

<p>Lacrimal gland tissue from IL-1α injected mice were double-stained for muscle related proteins desmin and β-taxilin. Panels represent staining from 2 days post IL-1α injected lacrimal glands which were counterstained with DAPI to visualize cell nuclei. Arrows represent cells expressing both proteins; arrowheads represent cells expressing β-taxilin only; stars represent cells expressing desmin only; BV represents a blood vessel. Scale bars represent 25 μm.</p

Mass cytometry (CyTOF immunophenotyping) antibody panel.

<p>Mass cytometry (CyTOF immunophenotyping) antibody panel.</p

RNA-Seq and CyTOF immuno-profiling of regenerating lacrimal glands identifies a novel subset of cells expressing muscle-related proteins

<div><p>The purpose of the present studies was to use CyTOF and RNA-Seq technologies to identify cells and genes involved in lacrimal gland repair that could be targeted to treat diseases of lacrimal gland dysfunction. Lacrimal glands of female BALB/c mice were experimentally injured by intra-glandular injection of interleukin 1 alpha (IL-1α). The lacrimal glands were harvested at various time points following injury (1 to 14 days) and used to either prepare single cell suspensions for CyTOF immuno-phenotyping analyses or to extract RNA for gene expression studies using RNA-Seq. CyTOF immuno-phenotyping identified monocytes and neutrophils as the major infiltrating populations 1 and 2 days post injury. Clustering of significantly differentially expressed genes identified 13 distinct molecular signatures: 3 associated with immune/inflammatory processes included genes up-regulated at days 1–2 and 3 associated with reparative processes with genes up-regulated primarily between days 4 and 5. Finally, clustering identified 65 genes which were specifically up-regulated 2 days post injury which was enriched for muscle specific genes. The expression of select muscle-related proteins was confirmed by immunohistochemistry which identified a subset of cells expressing these proteins. Double staining experiments showed that these cells are distinct from the myoepithelial cells. We conclude that experimentally induced injury to the lacrimal gland leads to massive infiltration by neutrophils and monocytes which resolved after 3 days. RNAseq and immunohistochemistry identified a group of cells, other than myoepithelial cells, that express muscle-related proteins that could play an important role in lacrimal gland repair.</p></div

Principal component and heatmap analyses of RNA-sequencing samples.

<p>All samples were quality controlled to identify samples for exclusion from differential expression analysis. (A)Principal components analysis (PCA) was used to investigate replicate variability with 3 distinct clusters successfully identified. (B) Heatmap analysis was also used to cluster samples to identify samples for exclusion which identified 4 distinct clusters of samples. Samples (arrows in A, red circles in B) outside the defined clusters were excluded from downstream analyses.</p

Representative molecular signatures of lacrimal gland inflammation and repair.

<p>Expression (log2[fold change]) pattern of clusters representative of the 8 consolidated clusters were plotted for days 1, 2, 3, 4, 5, and 7. Threshold for significant up/down-regulation (+/- 1 = log2[+/-2]) is indicated by dotted blue line.</p

Differential expression of collagen chains by day.

<p>Differential expression of collagen chains by day.</p

Expression pattern of significantly differentially expressed genes by day.

<p>The significantly differentially expressed genes at days 1, 2, 4, and 5 were plotted to visualize the global expression of these genes across all time points (days 3, 7, 14 and 14S data not plotted based on the small number of genes). Day 4 and 5 significantly differentially expressed genes exclude day 1 and 2 expression for visualization purposes (very few genes up/down-regulated). Data points colored based on regulation at the day of interest; green = strongly up-regulated, red, up-regulated, orange = strongly down-regulated, blue = down-regulated.</p