CTGF AND CYR61 EXPRESSION IN NORMAL WISTAR RAT GINGIVAL TISSUES (Ab- stract): Sometimes, the term growth factor is used in parallel with the term ...
Revista Românã de Anatomie funcþionalã ºi clinicã, macro- ºi microscopicã ºi de Antropologie CTGF and Cyr61 Expression in Normal Wistar Rat Gingival Tissues ORIGINAL PAPERS Vol. X Nr. 2 2011
CTGF AND CYR61 EXPRESSION IN NORMAL WISTAR RAT GINGIVAL TISSUES Mihaela Mitrea1, L.L. Francu2, Dorelia Lucia Calin3 1. Tutor, Department of Anatomy 2. Assistant professor, Department of Anatomy 3. Assistant, Department of Cariology and Odontotherapy Iasi Gr. T. Popa University of Medicine and Pharmacy CTGF AND CYR61 EXPRESSION IN NORMAL WISTAR RAT GINGIVAL TISSUES (Abstract): Sometimes, the term growth factor is used in parallel with the term cytokine. Historically, cytokines have been associated with the hematopoietic and immune system cells. CCN protein family has six multifunctional members. CTGF is a matricell factor that collaborates with other growth factors, growth factor receptors, extracellular matrix and its receptors. Cyr61 protein promotes cell proliferation, chemotaxis, angiogenesis, and cell adhesion. Material and method: The study was carried out on 4 series of 5 adult, male Wistar rats each, weighing 260±23 g. The positive reactions were observed with a Nikon microscope and consisted in the occurrence of a brown precipitate with cytoplasmic pattern corresponding to the location of the monitored molecules. Results and discussions: The results regarding CTGF expression revealed an extremely wide variation in the presence of this molecule, 4 of 5 rats presenting staining only at the level of some capillary endothelia in lamina propria and not in the gingival epithelium. Cyr61 expression in the investigated series partially overlaps that of CTGF. Conclusions: CTGF molecule expression greatly varies within the same series. Cyr61 expression partially overlaps CTGF expression. This heterogenicity within the same animal series demonstrates that the occurring tissue perturbations might be of different degrees, probably due to some individual peculiarities. Key words: CTGF, Cyr61, GROWTH AFCTORS, GINGIVAL TISSUES
INTRODUCTION Sometimes, researchers use in parallel with the term growth factor the term cytokine. Historically, cytokines have been associated with the hematopoietic and immune system cells (1, 2). The term growth factor refers to a protein with the ability of stimulating cell growth, proliferation and differentiation. Growth factors are important because they regulate a multitude of cellular processes (3). Growth factors act like signaling molecules between cells, and often initiate differentiation and maturation, processes that vary with the growth factor (4). While growth factors have a positive effect on cell division, cytokine is neutral with regard to where the molecule affects proliferation (4, 2). CCN protein family has six multifunctional members, including CCN1 (Cyr61), CCN2 (connective tissue growth factor - CTGF), CCN3
(Nov), CCN4 (WISP1), CCN5 (WISP2), and CCN6 (WISP3) (5). These factors have a highly preserved structure, which consists in four preserved cysteine-rich fields (6). The biological function of this protein family includes the up and downregulation of connective tissue cells proliferation and differentiation and the regulation of extracellular matrix accumulation. CTGF is found in endothelial and granular cells, fibroblasts, mesangial cells, chondrocytes and osteoblasts, and also in biological tissues and fluids in low molecular mass forms that contain domains 3 and 4 that prevent the mitogenic activity (7, 5). Moreover, recent studies show that CTGF links to other growth factors inhibiting or stimulating their activity (8). All reviewed studies support the idea that CTGF is a matricell factor that acts together with other growth factors, growth factor receptors, extracellular matrix and its receptors (9). 183
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Cyr61 protein promotes cell proliferation, chemotaxis, angiogenesis, and cell adhesion. This protein seems to play an important role in wound healing (at the level of skin fibroblasts), and expression of a great number of genes involved in angiogenesis, inflammation and extracellular matrix remodeling, including VEGA-A, VEGA-C (basic protein), MMP1 (metalloproteinase matrix 1), MMP3 (metalloproteinase matrix 3), TIMP1 (tissue metalloproteinase inhibitor), and cell proliferation trough a-v/ß-3 integrins (10, 11). Cyr61 and CTGF have an almost similar structure, but the initiation of extracellular matrix production or accumulation seem to be CTGF-specific (12). MATERIAL AND METHOD The study was carried on 4 series of 5 male, adult Wistar rats (Rattus novergicus albinus) weighing 260±23 g. Series I was designed to investigate CTGF expression, series II Cyr61 expression, series III NFkB expression, and series IV as controls. All animals were kept in the same conditions with free access to food and water for 3 months. The animals were killed by intraperitoneal administration of a dose of sodium thiopental. The mandible together with the two lower incisors and the surrounding gingival tissues were collected from each animal. Immediately after that the tissues have been prepared for microscopy. To immunohistochemically investigate the tissue section mounted on a slide, the paraffin traces that prevent the access of detection antibodies to the epitopes in view had to be removed. The samples were then subjected to antigenic retrieval by incubation in retrieval solution (Dako Retrieval Solution, Dako Glostrup, Denmark), pH 6.1, for 20 minutes at 980C (water bath). Endogenous peroxidase were blocked with Dual Endogenous Enzyme Block (reactive present in the Dako En Vision+Dual Link System-HRP (DAB+) kit), 5 minutes at room temperature (RT), followed by washing in distilled water, 5 minutes at RT and phosphate buffer solution (PBS), 5 minutes at RT. For labeling, rat anti-Cyr61 rabbit antibody (sc-13100, Santa Cruz Biotechnology) and rat anti-Cyr61 mouse antibody (sc-733869, Santa Cruz Biotechnology), both in 1:200 dilution were used. The specific reactions were detected with the Dako En Vision+Dual Link System HRP (DAB+) developing kit. The study proto184
col required the initial sample incubation with a secondary anti-rabbit and anti-mouse antibody associated with a polymer, in its turn coupled with peroxidase molecules (Labeled Polymer HRP), 30 minutes at RT. The enzyme was washed in PBS (phosphate buffered saline). The sections were incubated with 3,3diaminobenzidine (DAB), activated extemporaneously with 3% distilled water (H2O2) for 30 minutes at RT, counterstained with Mayers hematoxilin (Dako, Denmark) and mounted with Canada balsam. The negative controls, that is the absence of antigen-antibody link, were represented by sections from the same tissues incubated with rabbit and mouse antibodies of the same isotype (IgG) as the used primaries, but with irrelevant specifications (control IgG, Pharmigen, and mouse negative control, Dako, Denmark). The positive reactions, under the form of a brownish precipitate with cytoplasmic patter (CTGF, Cyr61) corresponding to the location of the monitored molecules, were seen with a Nikon microscope. These expressions were analyzed qualitatively. The presence of the molecules (growth factors) was judged by the identification of color signals, without estimating the percentage of positive cells (fibroblasts) or considering the intensity of staining as an antigen level determination. This is accounted for by the difficulties we were faced with during the investigation, starting with the difficulty in identifying the gingival tissues and cutting the blocks, and the frailty of the tissues that quite often became degraded. RESULTS Our data on CTGF expression have identified the presence of this molecule in the gingival tissues of the study rats. In the gums of series I rats there was an extremely high variation in the presence of the molecule. Thus, of the 5 rats only 4 did not presented staining in the gingival epithelium, but only at the level of some capillary endothelia in lamina propria (fig.1, fig. 2), while in but one rat in this series a weal signal was obtained in both gingival epithelium and endothelium (fig. 3). Cyr61 expression in our study animals partially overlaps that of CTGF, meaning that the epithelial and endothelial cells, but also a small number of fibroblasts are positive. The gingival epithelium of the animals was poorly positive
CTGF and Cyr61 Expression in Normal Wistar Rat Gingival Tissues
Fig. 1. CTGF staining (x 100). Series I rat
Fig. 2. CTGF staining (x 100)
Fig. 3. CTGF staining (x 100). Notice the weak signal in the epithelium (x200)
Fig. 4. Cyr61 molecule staining. Control series rat (x200)
or negative (fig. 4). However, the absence of signals in the extracellular matrix was noticed, suggesting that this growth factor has only an intracellular location, and thus differentiating it from CTGF. Cyr61 expression involves mainly the endothelial cells, as seen in all our study animals and in agreement with the data in the literature that support an autocrine intervention of Cyr61 on vascularization. The major effect of this action is the stimulation of endothelial growth and formation of new vessels, the direct consequence being cell growth at this level (epithelial, endothelial, inflammatory and fibroblast).
stituents, such as type I collagen (13). In physiological situations, Cyr61 molecule is not detected in epithelial cells but only in endothelial cells, fibroblasts, and smooth muscle cells. In the series under study Cyr61 expression partially overlaps CTGF expression, and involves mainly the endothelial cells, as seen in all study rats. This finding is in agreement with the data in the literature suggesting an autocrine intervention of Cyr61 on vascularization (13, 14). The main effect of this action is the stimulation of endothelial growth and formation of new vessels, with cell growth at this level (epithelial, endothelial, fibroblasts and inflammatory cells) as a direct result.
DISCUSSIONS CTGF and Cyr61 molecules are cysteine-rich proteins belonging to the same growth factors family. These growth factors intervene in the regulation of apoptosis, angiogenesis, cell proliferation, and CTGF seems to stimulate the synthesis of extracellular matrix con-
CONCLUSIONS The presence of CTGF molecule in the gingival tissues of the study rats was identified. An extremely significant variation in the presence of this molecule in the gums of the investigated rats was found. Thus, 4 of 5 rats did not 185
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present staining in the gingival epithelium but only in some capillary endothelia of lamina propria, while in the remaining rat a weak signal in the gingival epithelium and endothelium was recorded. In the series under study Cyr61 expression partially overlaps CTGF expression, and in-
volves mainly the endothelial cells, as seen in all study rats. The heterogeneity in the expression of these two molecules even in the same animal series demonstrates that the occurring tissue perturbations might be different degrees, probably as a result of some individual particularities.
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