Sylvain L. Guérin

Reconstructed Human Cornea Produced in vitro by Tissue Engineering

The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson’s trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as β1, α3, α4, α5, and α6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4–5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the α4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.

Can we produce a human corneal equivalent by tissue engineering

Tissue engineering is progressing rapidly. Bioengineered substitutes are already available for experimental applications and some clinical purposes such as skin replacement. This review focuses on the development of reconstructed human cornea in vitro by tissue engineering. Key elements to consider in the corneal reconstruction, such as the source for epithelial cells and keratocytes, are discussed and the various steps of production are presented. Since one application of this human model is to obtain a better understanding of corneal wound healing, the mechanisms of this phenomenon as well as the function played both by membrane-bound integrins and components from the extracellular matrix have also been addressed. The analysis of integrins by immunohistofluorescence labelling of our reconstructed human cornea revealed that beta(1), alpha(3), alpha(5), and alpha(6) integrin subunits were expressed but alpha(4) was not. Laminin, type VII collagen and fibronectin were also detected. Finally, the future challenges of corneal reconstruction by tissue engineering are discussed and the tremendous applications of such tissue produced in vitro for experimental as well as clinical purposes are considered.

Characterization of wound reepithelialization using a new human tissue-engineered corneal wound healing model.

PURPOSE The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization. METHODS Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied. RESULTS Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that alpha v beta6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue. CONCLUSIONS The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.

Impact of Cell Source on Human Cornea Reconstructed by Tissue Engineering

PURPOSE To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. METHODS Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. RESULTS The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. CONCLUSIONS The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.

Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro

The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin α5β1. In this work, we determined whether the activity directed by the α5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/α5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the α5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3′ half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its α5β1 integrin activates a signal transduction pathway that results in the transcriptional activation of the α5 gene likely through altering the phosphorylation state of Sp1.

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

BackgroundPoly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI. In the present study, we examined whether suppression of PARP-1 expression in embryonic fibroblasts derived from PARP-1 knockout mice (PARP-1-/-) might alter the expression and/or DNA binding properties of Sp1 and NFI. We also explored the possibility that Sp1 or NFI (or both) may represent target proteins of PARP-1 activity.ResultsExpression of both Sp1 and NFI was found to be considerably reduced in PARP-1-/- cells. Co-immunoprecipitation assays revealed that PARP-1 physically interacts with Sp1 in a DNA-independent manner, but neither with Sp3 nor NFI, in PARP-1+/+ cells. In addition, in vitro PARP assays indicated that PARP-1 could catalyze the addition of polymer of ADP-ribose to Sp1, which also translated into a reduction of Sp1 binding to its consensus DNA target site. Transfection of the PARP-1 promoter into both PARP-1+/+ and PARP-1-/- cells revealed that the lack of PARP-1 expression in PARP-1-/- cells also results in a strong increase in PARP-1 promoter activity. This influence of PARP-1 was found to rely on the presence of the Sp1 sites present on the basal PARP-1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP-1-/- cells. Subjecting PARP-1+/+ cells to an oxidative challenge with hydrogen peroxide to increase PARP-1 activity translated into a dramatic reduction in the DNA binding properties of Sp1. However, its suppression by the inhibitor PJ34 improved DNA binding of Sp1 and led to a dramatic increase in PARP-1 promoter function.ConclusionOur results therefore recognized Sp1 as a target protein of PARP-1 activity, the addition of polymer of ADP-ribose to this transcription factor restricting its positive regulatory influence on gene transcription.

Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells

The cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) modulates cell cycle, apoptosis, senescence and differentiation via specific protein–protein interactions with the cyclins, cyclin-dependent kinase (Cdk), and many others. Expression of the p21 gene is mainly regulated at the transcriptional level. By conducting both ligation-mediated PCR (LMPCR) and chromatin immunoprecipitation (ChIP) in vivo, we identified a functional target site for the transcription factor, nuclear factor I (NFI), in the basal promoter from the p21 gene. Transfection of recombinant constructs bearing mutations in the p21 NFI site demonstrated that NFI acts as a repressor of p21 gene expression in various types of cultured cells. Inhibition of NFI in human skin fibroblasts through RNAi considerably increased p21 promoter activity suggesting that NFI is a key repressor of p21 transcription. Over-expression of each of the four NFI isoforms in HCT116 cells established that each of them contribute to various extend to the repression of the p21 gene. Most of all, over-expression of NFI-B in doxorubicin, growth-arrested HCT116 increased the proportion of cells in the S-phase of the cell cycle whereas NFI-A and NFI-X reduced it, thereby establishing a role for NFI in the cell cycle dependent expression of p21.

Electrophoretic Mobility Shift Assays for the Analysis of DNA-Protein Interactions

Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

Nuclear factor 1 interferes with Sp1 binding through a composite element on the rat poly(ADP-ribose) polymerase promoter to modulate its activity in vitro

Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and they share a structure common to housekeeping genes, as they lack a functional TATA box and contain multiple GC boxes, which bind the transcriptional activator Sp1. We have previously shown that, although Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely tuned modulation is likely dependent on other transcription factors that bind the rPARP proximal promoter in vitro. In this study, we identified one such factor as NF1-L, a rat liver isoform of the nuclear factor 1 family of transcription factors. The NF1-L site on the rPARP promoter overlaps one of the Sp1 binding sites previously identified, and we demonstrated that binding of both factors to this composite element is mutually exclusive. Furthermore, we provide evidence that NF1-L has no effect by itself on rPARP promoter activity, but rather down-regulates the Sp1 activity by interfering with its ability to bind the rPARP promoter in order to modulate transcription of the rPARP gene.

Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1.

We have previously shown that a trans-acting protein produced in some tissue culture cells positively control the transcriptional activity directed by the mouse p12 promoter. This nuclear protein exerts its positive activity by interacting with a regulatory sequence designated p12.A and located between the TATA and CCAAT box elements on the p12 gene promoter. Using DNase I and dimethyl sulfate methylation interference footprinting techniques coupled with gel retardation assays, we found evidence that the protein which binds to the p12.A element is the well-known transcription factor Sp1. Mutational analysis in transient transfection assays confirmed the positive activity exerted by this protein in every cell line tested. In agreement with this observation, we detected a p12.A-Sp1 binding activity in nuclear extracts prepared from all cell lines used. However, a similar binding activity could not be detected in a number of nuclear extracts prepared from normal mouse tissues. In this report, we provide the evidence that the lack of Sp1-binding activity results from the degradation of Sp1 in the kidney, liver, and pancreas of the mouse.