Isolation and Propagation of Mesenchymal Stem Cells from the Lacrimal Gland

The authors previously reported that the murine lacrimal gland contains stem/progenitor cells whose number increases during tissue injury and repair. In this study, mesenchymal stem cells were isolated and propagated from injured lacrimal glands

Role of cPKC␣ and nPKC in EGF-Stimulated Goblet Cell Proliferation

PURPOSE. The authors determined the role of the protein kinase C (PKC) isoforms cPKC␣ and nPKC in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells. METHODS. Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Gö 6983 (10 Ϫ10 -10 Ϫ7 M) for 20 minutes before stimulation with EGF (10 Ϫ7 M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKC␣ (Ad DNPKC␣, 10 4 pfu), dominant-negative nPKC (Ad DNPKC, 10 4 pfu), and wild-type cPKC␣ (Ad WTPKC␣, 10 7 pfu), and proliferation was measured. RESULTS. In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53% Ϯ 15%. In human goblet cells, EGFstimulated proliferation was completely inhibited by calphostin C. PKC␣, -␤I, -␤II, -␦, -, -/, -, -␥, and -were found in cultured rat goblet cells. Ad DNPKC␣ and Ad DNPKC inhibited EGF-stimulated proliferation in rat goblet cells by 78% Ϯ 6% and 92% Ϯ 8%, respectively. Incubation with Ad WTPKC␣ alone significantly increased proliferation. CONCLUSIONS. cPKC␣ and nPKC play key roles in conjunctival goblet cell proliferation. (Invest Ophthalmol Vis Sci. 2009;50: 614 -620

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

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

Quantification of immune cell populations.

<p>The number of cells per thousand of analyzed cells were plotted by day for (A) CD45+ (immune cells) CD45- (non-immune cells), (B) cells with large population change, monocytes and neutrophils, and (C) cells with small population change, natural killer (NK), B, plasmacytoid dendritic (pDC), CD4+ and CD8+ T cells. Insert shows representative immunofluorescence staining of Ly-6G (neutrophils) 1 day post IL-1α injection.</p

Venn diagram of overlap in significant gene regulation for each day.

<p>A Venn diagram of the differentially expressed genes at days 1, 2, 3, 4, and 5 was created to identify the overlap in gene expression.</p