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RESEARCH REPORT |
1 Department of Periodontology, Facultad de Odontologia, University of Sevilla, c/Avicena s/n, 41009 Sevilla, Spain;
2 Institute of Anatomy and Pathologic Histology, Università Politecnica delle Marche, Ancona, Italy;
3 Institute of Biochemistry, Università Politecnica delle Marche, Ancona, Italy;
4 Department of Medical Physiology and Biophysics, University of Sevilla, Spain; and
5 Emeritus Professor of Periodontology and Preventive Dentistry, University of London, UK
* corresponding author, pbullon{at}us.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: nifedipine • cyclosporin A • gingival overgrowth • Fura • intracellular calcium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Fibroblast metabolism is essential in collagen turnover and implicates intracellular calcium leading to gingival overgrowth (McCulloch, 2004). Cellular signaling mechanisms may be involved in the metabolic status change due to drugs, in particular those modulating intracellular calcium ion concentration, [Ca2+]i (Barclay et al., 1992; Hallmon and Rossmann, 1999). With regard to the measurement of the cytoplasmic calcium concentration, the ratiometric method has become a standard, to avoid the interference of intracellular organelles leading to anomalous values of cytoplasmic calcium concentration, as determined by imaging techniques (Grynkiewicz et al., 1985). It has been demonstrated that nifedipine blocks the increase in intracellular calcium induced by the mechanical stretching of gingival fibroblasts (Arora et al., 1994). Also, cyclosporin inhibits interleukin-2-dependent T-cell proliferation by lowering cytosolic concentrations of free calcium ions (Gelfand et al., 1987).
Given these data, we used an experimental rat model to analyze collagen levels and microvessel density in nifedipine and cyclosporin gingival biopsies. We then correlated these findings with the intracellular calcium concentration ([Ca2+ ]i) in gingival fibroblasts. We used fresh fibroblasts extracted from the gingival biopsies to explore the relationship between a change in the [Ca2+]i in fibroblasts and the induced morphological tissue features. Fresh culture of gingival cells allowed us to make reliable intracellular calcium determinations in quasi in vivo conditions.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Intracellular Calcium
Samples of the buccal gingiva of the lower right first molar were taken for the measurement of intracellular calcium. Gingival specimens were washed with an external solution [NaCl 124, KCl 5, MgSO4 1.3, NaH2PO4 1.2, NaHCO3 25, glucose 10, MgCl2 1.3, MOPS (Sigma, Hamburg, Germany) 25, CaCl2 2.4, all in mM], then immersed for 15 min in type IV collagenase (1 mg/mL, Sigma, Hamburg, Germany) (36.6°C, 99% relative humidity, 4.9% CO2 pressure). After being repeatedly washed with the external solution, cells were incubated for 45 min in Dulbecco’s modified Eagle medium (Sigma, Hamburg, Germany) under the same conditions, supplemented with fetal bovine serum (10%), L-glutamine (2 mM), and penicillin/streptomycin (1 mg/1 mL) (Sigma, Hamburg, Germany). The cells were dropped onto the coverslip at the bottom of the chamber for microscopic visualization, and incubated with 50 µL of a solution of Fura-2/AM (cell permeant Acetoxy-Methylesther) (5 mM) (Molecular Probes, Amsterdam, The Netherlands) containing 25% (v/v) dimethyl-sulfoxide (Sigma, Hamburg, Germany). The chamber was positioned on the stage of an inverted microscope equipped with fluorescence objectives (AXIOVERT-35-Zeiss, Göttingen, Germany) that allowed for the excitation of the dye at two wavelengths (340 and 380 nm), whereas the fluorescence emission of the specimen (510 nm) was recorded with a –40°C cooled CCD (Coupled Charge Device) 12-bit camera (Spectra Source, Los Angeles, CA, USA), located in a parallel port of the microscope, and data were stored in a computer. Every exposure was corrected for bias and dark current generated by the CCD according to the time required for image capture. We obtained the 340-nm and 380-nm ratio values of fluorescence images by dividing the images pixel by pixel after subtraction of any background fluorescence.
In vitro calibration of Fura-2 AM was performed in a 5-µL prism-shaped microcuvette filled with a solution containing Fura-2 pentapotassium (20 µM) in different pCa buffered solutions, obtained from Molecular Probes (Amsterdam, The Netherlands), and their calcium concentrations were checked with a calcium-selective microelectrode. Calibration curves at different fluorescence ratios were fitted according to the general expression: (Fmax-Fmin)/{1+Kdx10expX} + Fmin (Fmax, maximal value of fluorescence; Fmin, minimal value of fluorescence; Kd, dissociation constant). According to the 340/380 ratio, the Kd was 120 nM. Calcium concentrations were calculated according to the equation: [Ca2+] = Kdß(R-Rmin)/(Rmax-R) (Grynkiewicz et al., 1985), where R is the 340/380 ratio of the fluorescence intensity generated at these wavelengths in the cell, Rmax is the maximum ratio obtained at saturating calcium concentrations, and Rmin is the minimum ratio measured with the lowest calcium concentration. Kd is the apparent dissociation constant of FURA-2 for calcium, and ß is the ratio of the 380-nm fluorescence under minimum and maximum [Ca2+] conditions.
Histological Study
Mandibles were dissected and fixed in 10% formalin for histological study. The buccal gingiva of the lower left first molar was dissected and paraffin-embedded. For each animal, the most representative and oriented paraffin-embedded tissue block was selected for measurement of the marginal gingival width, collagen content, and microvessel density. The width of the marginal gingiva was measured on tissue sections stained with hematoxylin-eosin. The distance from the alveolar bone periosteum to the basal membrane underlying the squamous epithelium was measured on histological sections by a micrometric grid located in the ocular of a light microscope (Fig. 1
, arrows). Serial sections from the most representative tissue block were stained with the Picro-Mallory technique to highlight the content of collagen and to facilitate measurement of the proportion of the total stroma occupied by collagen. Measurements were effected on at least 30 microphotographs taken for each stained section at high magnification (400X). Camedia software (Olympus DP soft, Hamburg, Germany) was used for image acquisition and data elaboration. We assessed the microvessel density in the marginal gingival mucosa on sections stained with anti-CD34 antibody (clone HPCA1, Beckton Dickinson, Erembodegem, Belgium) by counting the number of CD34-positive small vessels under a light microscope at a magnification of 400X, covering an area of within 0.16 mm2 per field. Any brown-stained endothelial cell or endothelial cell cluster that was clearly separated from adjacent microvessels was considered a single countable microvessel. Microvessel density was then expressed as the number of counted microvessels per mm2. Measurements of the marginal gingival width, collagen content, and microvessel density were repeated 3x for each slide; the mean value was calculated and taken into account for further statistical analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 2). Collagen content expressed by area was highest in the nifedipine group, followed by the cyclosporin one. The differences were statistically significant (p < 0.0001), while the Bonferroni test showed differences among all 3 groups (Fig. 3).
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). By the deconvolution equation of the calibration curve for Fura-2, this ratio corresponded to a resting cytoplasmic calcium concentration of 130 ± 12 nM, while, for the cells of the control and nifedipine groups, the [Ca2+ ]i ranged between 50 and 90 nM. Data from spatial distribution of calcium-induced fluorescence in the control group, nifedipine group, and cyclosporin group cells (Fig. 4) showed an intracellular domain of calcium, possibly related to the in situ cell orientation in the gingival tissue.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of our data confirms the pro-angiogenetic properties of cyclosporin in gingival tissues, since some authors (Ayanoglou and Lesty, 1999) have previously suggested measuring the numbers of vessels/mm2 in semi-thin sections of the gingival connective tissue of rats treated with cyclosporin. However, other researchers found no increased numbers of vessels in rats treated with cyclosporin or nifedipine (Spolidorio et al., 2002), but they did not stain vessels by immunohistochemistry. Other authors found a decreased number of vessels immunostained with anti-CD34 in the renal peritubular capillaries of children affected by nephrotic syndrome after 1–2 yrs of cyclosporin treatment (Lim et al., 2004). The differences in angiogenetic response and fibrotic process related to nifedipine and cyclosporin may also be influenced by specific properties of different tissues, and the gingiva is exceptional for its extensive collateral microvasculature and relatively high collagen turnover rate. Such aspects should be considered in relation to side-effects in organ transplantation, such as in the kidneys, to evaluate possible relevant drug-induced influences during and after transplantation.
In our study, collagen seemed to be mainly responsible for gingival enlargement with nifedipine, more than angiogenesis. Therefore, we could not confirm previous data suggesting an increasing vascularization in nifedipine-treated rats in the chick chorioallantoic membrane (Dusseau and Hutchins, 1993). Our data, however, are in agreement with other results, which reported that nifedipine produced specific enhancement of synthesis of collagenous proteins by gingival fibroblasts (Henderson et al., 1997), and an increase of collagen in the myocardium of rats receiving nifedipine (Frolov, 2003). Further, when the amounts of gingival collagen in persons treated with cyclosporin, nifedipine, hydantoin, and a control group were compared, the area occupied by collagen was significantly greater in nifedipine than in the other pathology groups (Bonnaure-Mallet et al., 1995). In our study, the higher amount of collagen in the nifedipine group, as measured by the proportion of collagen in the total tissue, did not produce a homogeneous effect in gingival width, but the tissue was more fibrotic.
Cyclosporin has also been shown to produce an increase in the proportion of gingival collagen in humans (Wysocki et al., 1983). In fibroblasts treated with cyclosporin, significantly higher levels of expression of type I collagen have been demonstrated (Arzate et al., 2005). In our study, collagen increased in the cyclosporin group, but less than in the nifedipine one.
We hypothesized that a modification of [Ca2+ ]i in gingival fibroblasts might be among the underlying mechanisms for the differing patterns of gingival overgrowth in rats treated with nifedipine or cyclosporin. For these reasons, we used fresh gingival fibroblasts taken from the rats to explore the possible effects of a change in [Ca2+]i on other proliferative variables. Fresh culture of gingival cells allowed us to make reliable intracellular calcium determinations, depending on the preceding in vivo conditions. Chronic culture of gingival cell lines usually shows changes in [Ca2+ ]i not related to the in vivo location.
In our study, nifedipine seemed to have no influence on [Ca2+]i under resting conditions. However, the change in cytoplasmic calcium concentration during depolarization, induced by an increase in extracellular potassium, was less than that found in control cells. This finding agrees with the known low conductance of L-type calcium channels under resting conditions, and the blocking effects of nifedipine during the expected opening of these channels under depolarizing conditions. The intracellular calcium variations seen with nifedipine in rat cardiac fibroblasts with a Calcium Kit-Fluo3 (Dajindo Laboratories, Tokyo, Japan) did not show alterations in the fluorescence intensity of fluo 3 in the presence or absence of extracellular calcium (Yue et al., 2004). They also stated that nifedipine increased the gelatinolytic activity of matrix metalloproteinase (MMP-2) significantly in a dose-dependent manner. Analysis of our data supports these studies, indicating that there may be some other mechanisms for nifedipine to exert its effects on MMP-2 expression in fibroblasts, independently of intracellular calcium variation. The highest level of new collagen in the gingival tissue of nifedipine-treated animals has been ascribed to the low change in calcium concentration, in addition to the known effect of nifedipine on cathepsins B and L inhibition (Nishimura et al., 2002).
In contrast, the cyclosporin-treated cells in our study showed the highest cytoplasmic calcium concentration under resting conditions. Analysis of these data suggests that changes in intracellular calcium can regulate MMP dynamics underlying the gingival enlargement (Munshi et al., 2002). In effect, MMP-1 is an endopeptidase activated by divalent cations (Ca2+, Zn2+) and negatively regulated by TIMP (tissue inhibitor of metalloprotease), an endogenous inhibitor TIMP-2 that is co-expressed from fibroblasts and might be activated by Ca2+ (Goldberg et al, 1989). The latter would result in an inhibition of MMP underlying the gingival collagen overproduction in CsA-treated cells in which [Ca2+ ]i is increased. Another possible mechanism not considered in our study is the effect of CsA on the genetic expression of MMP and TIMP. It has been shown that CsA inhibits MMP-1 expression at both the mRNA and protein levels in a dose-dependent way. However, CsA had no effect on the TIMP-1 protein level, which is compatible with a decreasing activity of MMP-1 underlying the collagen proliferation in CsA-treated tissue (Hyland et al., 2003).
Cyclosporin is known to increase the permeability of liver plasma membrane to calcium, resulting in an increase in total cell calcium content (Nicchitta et al., 1985). Incubation of rat aortic vascular smooth-muscle cells with cyclosporin increased cytosolic calcium concentrations in response to several vasoconstrictor hormones (Lo Russo et al., 1997). The addition of cyclosporin to isolated rat renal proximal tubules caused a transient increase in intracellular calcium (Carvalho da Costa et al., 2003). In summary, our results support the hypothesis that the gingival overgrowth induced by nifedipine and/or cyclosporin treatment may be mediated by a resultant higher number of gingival vessels, and that changes in collagen metabolism are not mediated by intracellular calcium changes. However, intracellular calcium may play a role by direct or indirect inhibition of MMP activity and by decreasing collagen phagocytosis.
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ACKNOWLEDGMENTS |
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Received February 1, 2006; Last revision November 8, 2006; Accepted November 17, 2006
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REFERENCES |
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F/F. Changes in ratiometric values of fluorescence, thought to be proportional to intracellular free calcium, are shown in (d). The nifedipine group showed the lowest [Ca2+]i, but not significantly different from that in the control group. The cyclosporin group showed the highest cytoplasmic calcium concentration under resting conditions (*p < 0.05). The data are expressed as the mean ± SD.