N. Di Girolamo

Tracing the Fate of Limbal Epithelial Progenitor Cells in the Murine Cornea

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreERT2‐Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K‐14+ progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound‐healing, disease, and following transplantation. Stem Cells 2015;33:157–169

The collagenase-1 (MMP-1) gene promoter polymorphism - 1607/2G is associated with favourable prognosis in patients with colorectal cancer.

Matrix metalloproteinase (MMP) overexpression has been implicated in the pathogenesis of colorectal carcinoma (CRC). Accumulating evidence suggests that MMP promoter single nucleotide polymorphisms (SNPs) effecting gene transcription are associated with enhanced susceptibility for the development of malignant disease, increased tumour invasiveness and poor patient survival. The aim of the current investigation was to determine whether such associations exist in a large CRC patient/control study population. Using an allelic discrimination real-time polymerase chain reaction, polymorphisms in the MMP-1, MMP-2 and MMP-3 gene promoters (−1607, −1306, and −1612 bp, respectively) were assessed in normal blood mononuclear cells from patients with CRC (n=503) and control subjects (n=471). Genotypes corresponding to each MMP SNP were correlated with tumour characteristics and clinical outcome. The frequency of each genotype was not statistically different between patients and control subjects and no significant differences were noted between the genotypes and tumour characteristics for the three MMP SNPs. CRC patients with the 2G/2G genotype for the MMP-1 SNP had significantly better 5-year survival compared to patients with a 1G allele (P<0.05). Our results demonstrate that CRC patients with a 2G/2G genotype in the MMP-1 gene promoter SNP have a favourable prognosis. Although our results were unexpected, given that this genotype is associated with enhanced MMP-1 transcriptional activity, they are consistent with recent data highlighting the anti-tumorigenic properties of MMPs.

Neutrophil accumulation correlates with type IV collagenase/gelatinase activity in endotoxin induced uveitis

Background/aim: Anterior uveitis is a common inflammatory ocular disease characterised by protein accumulation and leucocyte infiltration in the anterior chamber. The aim of this study was to determine the expression of gelatinases in the aqueous humour (AH) and uvea in an animal model of endotoxin induced uveitis (EIU). Methods: EIU was established in Lewis rats following an intraperitoneal injection of lipopolysaccharide (LPS). AH and ocular tissue were obtained from control animals and those with EIU over a 1 week time course and the samples analysed immunohistochemically and by gelatin zymography. Results: Matrix metalloproteinase (MMP) 2 and 9 levels were elevated in rat AH over a 1 week time course. MMP-2 and MMP-9 levels peaked at the time of maximum uveal inflammation, before returning to baseline levels as the inflammation subsided. MMP-9 was detected in the latent and functionally active form. Total protein extracted from inflamed rat uveal tissue displayed no significant gelatinolytic modulation throughout the time course of EIU. Anterior chamber neutrophils and ciliary body epithelial cells were the most abundant source of the gelatinases. Conclusion: This study has revealed a correlation between infiltrating neutrophils and the presence of elevated gelatinases in EIU. The results suggest that these proteolytically active enzymes may be important mediators of the inflammatory response and contribute to matrix remodelling observed in uveitis. Furthermore, the excess production of MMPs may be a mechanism by which leucocytes, such as neutrophils, gain access to uveal tissue and AH. Therapeutic strategies aimed at reducing MMP activity may be of some benefit in the treatment of uveitis.

Human limbal epithelial progenitor cells express αvβ5-integrin and the interferon-inducible chemokine CXCL10/IP-10.

Stem cell (SC) therapy is the main treatment modality for patients with limbal stem cell deficiency. If limbal epithelial stem cells (LESC) can be more readily identified, isolated and maintained ex vivo, patients could be treated with better quality grafts. With prior knowledge that vitronectin (VN) is present within the LESC niche and that it supports LESC in vitro, we postulated that VN receptors (integrins αvβ3/5) are expressed by, and can be used to identify and isolate LESC. Immunolocalization studies were conducted on human corneas. Corneas were also used to expand limbal epithelial cells from either biopsies or enzyme-dissociated tissue and αvβ3/5 expression determined by flow cytometry. Integrin expressing cells were isolated by magnetic activated cell sorting then assessed by immunocytology, colony forming efficiency, RT-PCR and microarray analysis. Integrin αvβ5(+) cells co-localized to N-cadherin(+)/CK-15(+) putative LESC. αvβ5 was restricted to less than 4% of the total limbal epithelial cells, which expressed higher levels of CK-15 and formed more colonies compared to αvβ5(-) cells. Transcriptional profiling of αvβ5(+/-) cells by microarray identified several highly expressed interferon-inducible genes, which localize to putative LESC. Integrin αvβ5 is a candidate LESC marker since its expression is restricted to the limbus and αvβ5(+) limbal epithelial cells have phenotypic and functional properties of LESC. Knowledge of the niches molecular composition and the genes expressed by its SC will facilitate isolation and maintenance of these cells for therapeutic purposes.

Ultraviolet radiation-induced cytokines promote mast cell accumulation and matrix metalloproteinase production: potential role in cutaneous lupus erythematosus

Objective: To examine the role of mast cells (MCs), cytokines, and matrix metalloproteinases (MMPs) following ultraviolet B (UVB) radiation in cutaneous lupus erythematosus (CLE). Methods: Immunohistochemistry was used to determine the presence of MCs and the expression of MMP-1, MMP-9, interleukin (IL)-15, and CCL5/RANTES in skin from patients with CLE. Human keratinocytes were exposed to varying doses of UVB and supernatants were collected and assessed for IL-15, CCL5, MMP-1, and MMP-9 by protein assays. MC migration was determined against supernatants from UVB-treated keratinocytes. Results: MCs in the skin of patients with CLE were significantly increased. MMP-1 and MMP-9 expression was abundant in these lesions. Intense reactivity for IL-15 and CCL5 was found in skin, particularly in epidermal keratinocytes, from patients with CLE. UVB irradiation induced IL-15, CCL5, MMP-1, and MMP-9 production from keratinocytes in a dose- and time-dependent manner. Supernatants obtained from UVB-treated keratinocytes induced MC migration, which was attenuated by anti-IL-15 and anti-CCL5 neutralizing antibodies. IL-15 induced MC-derived MMP production. Conclusions: Our results indicate that MCs and MMPs may play a role in the skin inflammation in CLE. MC recruitment as well as MMP production may be perpetuated by UV irradiation through locally released mediators.

Functional Activity of Plasma Fibronectin in Patients With Diabetes Mellitus

Decreased wound healing and increased infection are major problems in patients with diabetes mellitus. Fibronectin plays a fundamental role in wound healing and acts as an opsonin for the phagocytosis of foreign antigens. The aim of this study was to ascertain the functional activity of plasma fibronectin from patients with diabetes mellitus. Initially, a modified Boyden chamber technique was used to measure cell migration on fibronectin purified from patients plasma and an enzyme-linked immunosorbent assay was used to measure the binding of gelatin. A sandwich assay was developed that enabled the capture of fibronectin directly from patients plasma without prior purification. With the use of a 96-well format, the binding of two different monoclonal antibodies could be compared simultaneously with the binding of gelatin and cell adhesion. In this way, differences in the function of particular domains of fibronectin from diabetic patients and control subjects could be measured. Results showed no difference between fibronectin from diabetic patients and control subjects with respect to the monoclonal antibodies binding in 1) the cell adhesion domain and 2) the heparin-binding domain. Furthermore, no detectable differences were noted with respect to cell adhesion, cell migration, or gelatin binding. These results suggest that diabetic patients receiving insulin treatment show no modulation of plasma fibronectin function, despite raised levels of circulating glucose.

The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities

Aims To investigate the effects of conditioned media from mesenchymal stem cells (MSC) on the wound healing activities of corneal stromal fibroblasts. Methods Cell cycle analysis and early stage activation of apoptosis, chemotactic chambers and fibroblast-populated type I collagen gels were used to assess corneal stromal fibroblast proliferation, migration and contraction, respectively. Fibroblasts were obtained from human donor corneas and MSC from fresh rat bone marrow. MSC conditioned media and fibroblast culture medium (FCM), with and without calf serum supplementation, were compared. Results MSC conditioned media and serum-free FCM had an inhibitory effect on the progression of corneal fibroblasts through the cell cycle. There was a significant increase in the number of cells in the G0–G1 phase for MSC conditioned media and serum-free FCM (p=0.001, p=0.97 respectively). Fibroblast migration and relaxed and stressed gel contraction were significantly inhibited by MSC conditioned media and serum-free FCM compared with FCM with serum (all p=0.001). Glucose and lactate analysis confirmed that these factors were not contributing to this effect. Conclusion MSC conditioned media was found to inhibit the wound healing activities of corneal stromal fibroblasts in vitro. Putative factors secreted by MSC could be developed for therapeutic use in corneal repair.

Ultraviolet B irradiation selectively increases the production of interleukin-8 in human cord blood-derived mast cells.

UVB irradiation modulates immune responses in the skin and is a major cause of sunburn, during which neutrophils accumulate in the skin. Because of their abundance in skin and ability to produce a variety of proinflammatory mediators, we propose that mast cells may play a key role in ultraviolet B (UVB)‐induced skin inflammation. Cord blood‐derived human mast cells were treated in vitro with varying doses of UVB and production of multiple cytokines was measured in culture supernatants. UVB exposure significantly increased the release of interleukin (IL)‐8 and modestly increased IL‐1α production, but cytokines such as IL‐2, IL‐4, IL‐6, IL‐10, IL‐12, IL‐13, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were unaffected. Cycloheximide reduced the UVB‐mediated induction of IL‐8 by 30–40%, suggesting that new protein synthesis contributed to IL‐8 production. In line with this, UVB treatment of mast cells significantly increased IL‐8 mRNA. In contrast to its effect on IL‐8 production, optimal doses of UVB did not provoke histamine or tryptase release, indicating little effect on degranulation. Our data suggest that mast cells may play a major role during UVB‐induced acute inflammation by selectively inducing cytokines involved in neutrophil recruitment.

Expression and distribution of matrix metalloproteinases and their inhibitors in the human iris and ciliary body

Aim: To determine the expression and distribution of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the normal human iris and ciliary body. Methods: Seven postmortem human eyes were fixed with formalin. The iris and ciliary body were dissected out and embedded in paraffin. The expression of MMPs -1, 2, 3, and 9, and TIMPs 1–4 in the iris and ciliary body was determined by a novel immunofluorescence technique and the results graded by masked observers. Results: Positive staining for MMPs and TIMPs was observed in all regions of the anterior uvea, and was more intense in the ciliary body than in the iris. Most MMPs and TIMPs showed similar patterns in their distribution. In the ciliary body, staining was strongest in the epithelium, and was localised to the epithelial cell cytoplasm, except for TIMP-3 which was strongly expressed in the basement membranes. In the iris, staining was most noticeable in the anterior border and anterior epithelial layer. Blood vessels in the stroma of the iris and ciliary body also stained moderately for MMPs and TIMPs. Conclusion: Both MMPs and TIMPs are widely expressed in the anterior uvea, with a positive correlation between their expressions. Their differential localisation in the ciliary body suggests they may have a role in maintaining homeostasis in the uveal tract.

Epidermal growth factor-like molecular species in normal bronchoalveolar lavage fluid.

Normal bronchoalveolar lavage fluid (BALF) contains mitogenic activity for fibroblasts and type 2 pneumocytes. A number of growth factors that might contribute to this activity have been identified in BALF. We found that a molecule or molecules able to bind to epidermal growth factor (EGF) receptors on mouse lung fibroblasts were present in normal mouse BALF and could be blocked by an antiserum to mouse EGF. Receptor binding was partially blocked by preincubation with heparin, indicating a relationship to the heparin-binding subgroup of EGF-like growth factors. Heparin markedly enhanced the mitogenic activity of BALF for fibroblasts, but we were unable to establish whether the EGF-like molecule contributed to this activity. Immunoblotting using the anti-EGF serum identified a protein of Mr 88,000 in concentrated BALF. The cellular source and physiological role of this growth factor merit further investigation.