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RESEARCH REPORT |
1 Department of Cariology, Restorative Sciences, and Endodontics, and
2 Department of Oral Medicine, Pathology, and Oncology, University of Michigan School of Dentistry, 1011 N. University, Rm. 5211, Ann Arbor, Michigan, 48109-1078, USA; and
3 Department of Pediatric Dentistry, University of São Paulo, Bauru School of Dentistry, Bauru, São Paulo, 17012, Brazil;
*corresponding author, jenor{at}umich.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: angiogenesis • streptococci • endodontic • pulpitis • LTA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Bacteria that invade the dentin, as well as their products that diffuse through dentinal tubules, are involved in the pathogenesis of pulpitis (Love and Jenkinson, 2002). Gram-positive bacteria have been frequently identified in dentinal tubules of both carious and non-carious teeth (Love and Jenkinson, 2002). The adhesion of oral streptococci to the dentinal walls and intra-tubular growth might be facilitated by their ability to recognize and bind to collagen type I (Liu and Gibbons, 1990). Lipoteichoic acid (LTA) is an amphiphilic molecule consisting of a polyglycerolphosphate with a complex glycolipid group attached to it (Sleytr et al., 1988). LTA is produced in large quantities by cariogenic bacteria when sucrose is available (Rølla et al., 1980). LTA is anchored by hydrophobic forces to the cell membrane of Gram-positive bacteria such as streptococci. However, when bacteria are grown at a low pH, a significant proportion of the LTA can be exported to the extracellular matrix (Jacques et al., 1979), where it induces the expression of several inflammatory mediators (Ginsburg, 2002).
The dental pulp is a low-compliance tissue enclosed within rigid non-expandable dentinal walls (Matthews and Andrew, 1995). An increase in vascular density and permeability may become deleterious and contribute to irreversible pulp pathology, since the dental pulp has limited ability to relieve internal pressures (Heyeraas and Berggreen, 1999). It is known that lipopolysaccharides (LPS) from Gram-negative bacteria induce VEGF expression in macrophages (Sakuta et al., 2001), and in a mixed population of pulp cells (Matsushita et al., 1999). However, we do not know if LTA from Gram-positive bacteria has a role in the regulation of angiogenesis. The purpose of this in vitro study was to examine if streptococcal LTA is sufficient to induce VEGF expression in macrophages, odontoblast-like cells, undifferentiated pulp cells, or fibroblasts.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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ELISA
Forty thousand cells per well were seeded in 12-well plates and allowed to attach overnight. Culture medium containing 0-80 µg/mL Streptococcus mutans (S. mutans) LTA (Sigma Chemical Co., St. Louis, MO, USA), or 0-80 µg/mL Streptococcus sanguis (S. sanguis) LTA (Sigma) was added to triplicate wells per condition. The LTA preparations used here were obtained by phenolic extraction from S. mutans (DSM 20381) or S. sanguis (ATCC 10556), and contained < 0.4% and < 1% protein (determined by the Lowry Protein Assay), respectively, according to the certificate of analysis from Sigma. After 24 hrs, the conditioned medium was collected from each well, and VEGF expression was analyzed by ELISA (Enzyme linked immunosorbent assay, Quantikine Murine VEGF Kit, R&D Systems, Minneapolis, MN, USA) according to manufacturer’s instructions. Absorbance was read in a spectrophotometer (DV-20, Beckman, Fullerton, CA, USA) at a single wavelength of 450 nm, and the concentration of VEGF was calculated from the standard VEGF curves. Recombinant mouse VEGF164 (R&D Systems) was used as a positive control for ELISA. Three independent experiments were performed per cell type and condition.
Trypan Blue Exclusion Assay
We used Trypan blue assays to normalize the data obtained from ELISAs by the number of viable cells. Briefly, after the collection of the conditioned medium for ELISA, the cells were trypsinized and re-suspended in a solution containing 0.2% Trypan blue stain (Gibco). The total cell number and the number of necrotic cells were counted in a hematocytometer (Hausser Scientific, Horsham, PA, USA) in an optical microscope at 200x.
Semi-quantitative RT-PCR
To evaluate the effect of LTA on VEGF mRNA expression, we performed RT-PCR from cells exposed to LTA. MDPC-23, OD-21, fibroblasts, or macrophages were allowed to attach overnight, and were then exposed to 0-80 µg/mL LTA for 24 hrs. Cells were retrieved from plates and washed with Hanks’ Balanced Salt Solution (Gibco), and total RNA was extracted with an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to manufacturer’s instructions. We performed cDNA synthesis and PCR amplification in a single tube using, simultaneously, a mouse VEGF and a GAPDH primer set with Super Script one-step RT-PCR with Platinum Taq kit (Invitrogen, Carlsbad, CA, USA). The VEGF primers were designed for amplification of mRNAs corresponding to all three mouse VEGF isoforms, VEGF120, VEGF164, and VEGF188. The primers used here were: VEGF 5'-CTGCTCTCTTGGGTCCACTGG and VEGF 3'-CACCGGGTTGGGTTGTCACAT (Hovey et al., 2001). GAPDH (glyceraldehyde adenosine-phosphate dehydrogenase) was used as an internal control for loading and PCR amplification. The RT-PCR products were analyzed by electrophoresis on 1% agarose gels containing ethidium bromide. The density of the bands corresponding to VEGF mRNA was measured with the NIH Image 1.62 software, and normalized against the density of the bands for GAPDH. Data presented are representative of three independent experiments.
Statistical Analysis
The statistical analyses were performed by one-way analysis of variance (ANOVA) or Student’s t test with the statistical software SigmaStat 2.0 (SPSS, Chicago, IL, USA). The significance level of the data was determined at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), OD-21 (Fig. 1B), or fibroblasts (Fig. 1C) expressed higher baseline levels of VEGF expression than did untreated macrophages (Fig. 1D). To examine if LTA induces VEGF expression in these cells, we exposed them to 0.8-80 µg/mL LTA for 24 hrs, and then performed ELISAs from conditioned medium. LTA induced up to a nine-fold increase in VEGF protein expression in macrophages (Fig. 1D), a 2.4-fold increase in MDPC-23 (Fig. 1A), and a 1.6-fold increase in OD-21 (Fig. 1B), as compared with controls. In contrast, we did not observe a significant up-regulation of VEGF expression in fibroblasts (Fig. 1C). MDPC-23 and macrophages treated with LTA showed a dose-dependent increase in VEGF expression, and OD-21 cells showed up-regulated VEGF expression only when induced with the highest LTA concentration (80 µg/mL). To evaluate if the VEGF up-regulation observed in MDPC-23 and OD-21 was specific to induction by S. sanguis LTA, we exposed these cells to S. mutans LTA and performed ELISAs. We observed that S. mutans LTA induced a trend of VEGF up-regulation in MDPC-23 (Fig. 2A) and OD-21 (Fig. 2B), but the fold induction of VEGF was lower compared with what we observed with S. sanguis LTA.
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). Analysis of these data suggests that the primary regulatory level for LTA-induced VEGF expression in the cells evaluated here is post-transcriptional.
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). Yet, both LTA and LPS induced VEGF expression in macrophages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A pathological increase in vascular leakage leads to edema and swelling, and causes severe complications in conditions such as sepsis syndrome, brain tumors, and also in inflammatory conditions such as rheumatoid arthritis (Thurston et al., 2000). VEGF was initially characterized as vascular permeability factor, due to the observation that its overexpression invariably results in edema (Senger et al., 1983). We know now that the ability of VEGF to enhance vascular permeability is estimated to be 50,000 times higher than that of histamine (Shulman et al., 1996). Previous reports have de-emphasized the role of LTA in the pathogenesis of acute infectious conditions such as sepsis syndrome (Ginsburg, 2002). This has been based on the observation that nanogram amounts of LPS are necessary to stimulate macrophages to synthesize pro-inflammatory cytokines, while amounts in the range of milligrams of LTA are required to cause similar responses (Ginsburg, 2002). Interestingly, about half of the cases of sepsis syndrome are caused by Gram-positive bacteria (Horn et al., 2000). Here, we observed that the stimulation of macrophages with 0.8 µg/mL LTA was sufficient to induce a four-fold induction of VEGF expression in these cells. This induction of VEGF expression by LTA is comparable with the induction that we observed when the same cells were stimulated with LPS (Botero et al., 2003). Analysis of these data suggests that the pro-angiogenic and pro-vascular permeability inputs induced by LTA and LPS have similar intensities. Since increased vascular permeability and edema are important components of sepsis syndrome, one speculates that LTA-induced VEGF expression by macrophages plays an important role in the pathogenesis of this syndrome when its etiological agents are Gram-positive bacteria. Analysis of our data also suggests that fairly low concentrations of LTA from Gram-positive bacteria might be sufficient to stimulate macrophages in the dental pulp to secrete VEGF, which in turn may result in edema, increased intra-pulpal pressure, and pain.
A recent report has suggested that blockade of VEGF signaling might be useful for treatment of patients with stroke (Paul et al., 2001). The rationale for this intervention is based on the observation that a significant component of the damage to neural tissue in these patients is because the hypoxia generated by cerebral ischemia stimulates VEGF expression that results in enhanced vascular permeability. Since the brain is a low-compliance organ encapsulated within rigid walls, the enhanced permeability and edema caused by VEGF expression result in an increase in intra-cranial pressure and exacerbate tissue damage. Similarly, the pulp is a highly vascularized tissue confined within non-expandable dentin walls. It is well-established that the dental pulp has a functional lymphatic system (Marchetti and Poggi, 2002), but its ability to relieve internal pressures might be hindered by the fact that the same foramen is used for both blood supply and fluid drainage (Heyeraas and Berggreen, 1999). It was recently demonstrated by immunohistochemistry that the expression of VEGF is strongly positive in cells constituting the inflammatory infiltrate of teeth with irreversible pulpitis (Artese et al., 2002). Here, we observed that LTA from Gram-positive bacteria induces VEGF expression, and that macrophages, odontoblasts, and undifferentiated pulp cells are effector cells for this response. We are currently undertaking experiments to evaluate the effect of blockade of VEGF signaling pathways on the survival of pulp cells on teeth with experimental pulpitis.
LTA shares many of its pathophysiological properties with LPS, a powerful pro-inflammatory agonist (Ginsburg, 2002). Treatment of macrophages with LPS has been shown to induce a distinct profile of gene expression that primes this cell to interact with its environment and to organize an immune response (Nau et al., 2002). Activated macrophages become polarized to facilitate the processes of secretion of cytokines, growth factors, and enzymes. The macrophage activation program is also induced by LTA from Gram-positive bacteria (Nau et al, 2002). LTA was shown to signal through CD14 and Toll-like receptors (Dziarski et al., 2000), which are also responsible for LPS-initiated signaling events (Guha and Mackman, 2001). Interestingly, LTA did not induce morphological changes in macrophages that could be observed with an optical microscope. Yet, LTA induced up to a nine-fold VEGF up-regulation in these cells. Analysis of these data, taken together, suggests that LTA-induced VEGF synthesis is not dependent upon changes in macrophage morphology, such as the ones observed when these cells are exposed to LPS (Botero et al., in press).
In the present study, we have demonstrated that streptococcal LTA induces VEGF expression in macrophages and pulp cells. A recent report has demonstrated that VEGF is expressed in the dentin matrix, and has suggested that its slow release from the matrix after injury might be beneficial to the reparative processes of the dentin-pulp complex (Roberts-Clark and Smith, 2000). Since VEGF is an important inducer of angiogenesis during the proliferative phase of wound healing (Nissen et al., 1998), its slow release from the dentin matrix may contribute to pulp repair after injury. In contrast, a rapid increase in VEGF expression mediated by pulp cells acutely exposed to bacterial toxins may ultimately result in pulp necrosis due to an increase in intra-pulpal pressure caused by VEGF-induced neovascularization and edema. The understanding of the molecular mechanisms underlying the regulation of neovascularization and vascular permeability observed in teeth with pulpitis may contribute to the development of therapeutic strategies designed to control these responses and prevent pulp necrosis.
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ACKNOWLEDGMENTS |
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Received September 3, 2002; Last revision January 22, 2003; Accepted February 27, 2003
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