- Email: [email protected]
Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts
Basic Research—Biology
Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Ikhlas A. El karim, BDS, PhD, Gerard J. Linden, BDS, BSc, PhD, Chris R. Irwin, BSc, BDS, PhD, and Fionnuala T. Lundy, BSc, PhD Abstract Introduction: Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro. Methods: An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts. Results: Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro. Conclusions: The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair. (J Endod 2009;35:829–833)
Key Words Angiogenesis, dental pulp, growth factors, neuropeptides
From the Department of Restorative Dentistry, School of Dentistry, Royal Group of Hospitals, Belfast, United Kingdom. Address requests for reprints to Dr Ikhlas A. El karim, Department of Restorative Dentistry, School of Dentistry, Royal Group of Hospitals, Grosvenor Rd, Belfast BT12 6BP, United Kingdom. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2009 American Association of Endodontists. doi:10.1016/j.joen.2009.03.005
A
ngiogenesis is a complex multistep process of new blood vessel formation that is regulated by numerous growth factors. In the dental pulp angiogenesis is crucial for tooth development and a prerequisite for successful repair after injury and inflammation (1). Angiogenic growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF-2), and transforming growth factor (TGF-b) have been identified in human dental pulp and dentin matrix (2–4). The dental pulp is a highly innervated tissue, and pulpal nerves, through the release of neuropeptides, have been shown to play an important role in inflammation (5). Neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) are up-regulated in dental pulp from carious teeth, suggesting a role for these neuropeptides in pulpal inflammation and healing (6–9). Neuropeptides have also been implicated in mediating angiogenesis (10), and given that fibroblasts express neuropeptides (11, 12) and neuropeptide receptors (12, 13), it is possible that neuropeptides might regulate angiogenic growth factor expression by pulpal fibroblasts. Indeed, mechanical injury to pulpal fibroblasts has been shown to be associated with angiogenic growth factor release (14). The aims of this study were therefore to establish the levels of expression of a range of angiogenic growth factors by pulp fibroblasts in vitro and to determine the effects of SP, CGRP, NPY, and VIP on expression levels after 24 and 48 hours in culture.
Materials and Methods Cell Culture Human pulp fibroblasts were derived from extracted healthy molar teeth collected with consent under the approval of the Office for Research Ethics Committees Northern Ireland as previously described (12). Fibroblasts were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere of 5% CO2, 95% air. On reaching confluence, fibroblast populations were propagated at a 1:3 split ratio. Cells between passages 4 and 5 were used in this study. Cell Treatment with Neuropeptides Pulp fibroblasts were seeded in 6-well plates at a density of 3 105 cells/mL and grown to confluence. Cells were rendered quiescent by culturing in DMEM supplemented with 1% FCS for 24 hours before stimulation with neuropeptides. A concentration of 1 10–7 mmol/L (15, 16) of NPY, VIP, CGRP, and SP (Bachem, Weil am Rhein, Germany) was added to the cells (in 1% culture medium) and incubated at 37 C for either 24 or 48 hours. All studies were undertaken in triplicate, and the supernatants from each culture condition were pooled and stored at –80 C until array analysis. Pulp fibroblasts in 1% culture medium (without neuropeptide treatment) were used as controls. Angiogenic Array Quantification of angiogenic cytokines produced by pulpal fibroblasts was performed by using the Quantibody Human Angiogenesis Array (RayBiotech, Inc, Norcross, GA) to determine the concentration of 10 cytokines simultaneously: angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), bFGF, heparin-binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), leptin, platelet-derived
JOE — Volume 35, Number 6, June 2009
Regulation of Angiogenic Growth Factor Expression by Neuropeptides
829
Basic Research—Biology growth factor (PDGF-BB), placental growth factor (PIGF), and vascular endothelial growth factor (VEGF). Each cytokine was arrayed in quadruplicate, together with positive and negative controls. Cytokine standards at predetermined concentrations were used to generate a 5-point standard curve. Before addition of standard cytokines or sample, the glass chip was blocked by incubation with 100-mL sample diluent for 30 minutes. The cytokine standards or pulp fibroblast supernatant (100 ml) were added and incubated at room temperature for 1 hour. After a washing step, 80 mL of detection antibody was added (1 hour at room temp) followed by a further washing step before addition of 80 mL of Alexa Fluor 555–conjugated streptavidin for 1 hour. Fluorescent signals were visualized with a laser scanner (Axon GenePix; Molecular Devices, Sunnyvale, CA) set at 555-nm excitation, 565-nm emission, and 10-mm resolution. Data were extracted and analyzed with RayBio Q Analyzer software (RayBiotech, Inc).
Results Angiogenic Growth Factor Expression by Pulp Fibroblasts In Vitro The human angiogenic growth factors angiogenin, ANG-2, EGF, bFGF, HB-EGF, HGF, leptin, PDGF-BB, PIGF, and VEGF were detected in pulp fibroblast supernatant from control cultures at both 24 and 48 hours (Figs. 1 and 2). After 24 hours, VEGF (2923 pg/mL) and angiogenin (3626 pg/mL) were expressed at high concentrations in pulp fibroblast supernatants (>1000 pg/mL), whereas EGF (0.4 pg/mL) and PDGF-BB (3.1 pg/mL) were much less abundant (<5 pg/mL). Several angiogenic factors tended to increase after 48 hours in control cultures (angiogenin increased from 3626 to 4263 pg/mL; HGF increased from 843 to 1396 pg/mL; PIGF increased from 63 to 194 pg/mL; VEGF increased from 2923 to 3744 pg/mL), whereas others remained unchanged (EGF) or were considerably less abundant at 48 hours compared with 24 hours (ANG-2 decreased from 61 to 15 pg/mL; leptin decreased from 371 to 69 pg/mL). Angiogenic Growth Factors Substantially Increased or Decreased by Neuropeptides A neuropeptide was considered to have induced a substantial change in growth factor expression if the increase was 50% greater than the control value, or if the decrease was 50% less than the control value. The effects of individual neuropeptides on growth factor expression fell into 1 of 3 groups: (1) substantial change (either greater than or less than 50% of control values) in growth factor expression (Fig. 1), (2) no change in growth factor expression (Fig. 2), or (3) variable effects on growth factor expression (Fig. 2). CGRP and VIP induced substantial increases in the expression of PIGF after 24 hours, which dropped below control values at 48 hours (Fig. 1A). CGRP had similar effects on HGF, with levels increased above 50% of control values at 24 hours but dropping below control values at 48 hours (Fig. 1B). All the neuropeptides tested had similar inhibitory effects on HBEGF after 24 and 48 hours of treatment (Fig. 1C). VIP and SP substantially decreased EGF levels after 48 hours of treatment, whereas NPY and CGRP effects were within 50% of control values (Fig. 1D). NPY was the only neuropeptide to produce inhibitory effects on PDGF-BB at both 24 and 48 hours, whereas the other neuropeptides produced no substantial effects (Fig. 1E). Angiogenic Growth Factors Not Altered by Neuropeptides Of the 4 neuropeptides studied, none altered the levels of angiogenin or VEGF beyond greater than or less than 50% of control values (Fig. 2A, B). 830
El karim et al.
Angiogenic Growth Factors Altered to Various Extents by Neuropeptides All 4 neuropeptides had variable effects on bFGF, ANG-2, and leptin. NPY had inhibitory effects on ANG-2 and leptin at 24 hours, but levels were substantially increased to higher than control values after 48 hours of treatment (Fig. 2C, D). A similar effect was observed with bFGF, with levels substantially lower than control at 24 hours but increased to just above control after 48 hours of NPY treatment (Fig. 2E). Although SP decreased leptin levels just below 50% of control values at 24 hours, leptin levels were increased just above 50% of control values at 48 hours (Fig. 2D). Similarly, CGRP increased leptin levels just above 50% of control values at 48 hours (Fig. 2D). CGRP induced a substantial increase in the expression of bFGF at both 24 and 48 hours. However, NPY and VIP decreased bFGF levels substantially at 24 hours, with levels returning to just above control values at 48 hours (Fig. 2E).
Discussion The dental pulp is highly vascularized connective tissue with good healing potential after injury and inflammation. In part, the healing potential is related to the ability of pulp cells to secrete growth factors that are essential for cell differentiation and neovascularization. The present investigation demonstrated for the first time that angiogenin, leptin, ANG-2, PDGF-BB, and HB-EGF are synthesized and secreted by pulp fibroblasts in vitro. Angiogenin was the most abundant growth factor secreted by pulp fibroblasts; its level exceeded that of VEGF. Furthermore, the levels of all 4 newly identified angiogenic factors in the dental pulp were higher than bFGF levels, suggesting potential physiologic roles in the dental pulp. The expression of VEGF, bFGF, PIGF, EGF, and HGF by pulp fibroblasts in vitro was confirmed, supporting previous work (4, 17, 18). The novel pulp fibroblast-derived molecules (angiogenin, ANG-2, HB-EGF, leptin) identified in the current study are potent angiogenic growth factors. Angiogenin and ANG-2 have essential roles in endothelial cell proliferation (19, 20). In the presence of VEGF, ANG-2 promotes proliferation and migration of endothelial cells and stimulates sprouting of new blood vessels. Angiogenin is crucial for bFGF-stimulated and VEGF-stimulated angiogenesis (21). HB-EGF is a potent inducer of tumor growth and angiogenesis (22), and leptin has proangiogenic effects, including induction of neovascularization and formation of capillary-like structures (23, 24). This study also demonstrated for the first time the involvement of neuropeptides in regulating angiogenic growth factor expression by pulp fibroblasts. Key angiogenic factors such as HGF and PIGF were up-regulated after 24-hour exposure to neuropeptides, whereas other factors (HB-EGF, PDGF-BB, and EGF) were decreased, and some remained unchanged (angiogenin, VEGF). These findings are in agreement with previous reports (25) and suggest a complex role for neuropeptides in regulating angiogenesis and healing. The up-regulation of neuropeptides after pulpal injury and inflammation has been well-established (7, 8, 26). During pulpal repair an angiogenic response is essential for healing and reparative dentin formation (27). A role for CGRP in pulp healing and dentin bridge formation after rat molar pulpotomy has been previously suggested (28). Our results suggest that CGRP might have a role in the early stages of angiogenesis, as evidenced by the induction of HGF, PIGF, and bFGF by pulp fibroblasts after 24 hours of treatment. SP has previously been shown to enhance endothelial cell proliferation and angiogenesis in vivo (29, 30). Although SP increased EGF expression (and to lesser extent bFGF) at 24 hours, it had a tendency to inhibit expression of many of the other angiogenic factors, suggesting a degree of complexity in the
JOE — Volume 35, Number 6, June 2009
A 350
B
Concentration of PIGF (pg/ml)
Concentration of HGF (pg/ml)
Basic Research—Biology
300 250 200 150 100 50
2500
2000
1500
1000
500
0
0 24
48
24
C
D 25
0.7
Concentration of EGF (pg/ml)
Concentration of HB-EGF (pg/ml)
48
Hours
Hours
20
15
10
5
0
24
0.6 0.5 0.4 0.3 0.2 0.1 0
48
24
Hours
48
Hours
Concentration of PDGF-BB (pg/ml)
E
5 Control
4.5
>50%
4
< 50%
3.5
NPY 3
SP
2.5
VIP
2
CGRP
1.5 1 0.5 0 24
48
Hours
Figure 1. Pulp fibroblast angiogenic growth factor expression substantially altered by neuropeptides: (A) PIGF, (B) HGF, (C) HB-EGF, (D) EGF, (E) PDGF-BB.
role of SP in angiogenesis. VIP also had variable effects, inhibiting HBEGF and PDGF-BB at 24 hours but increasing PIGF at the same time point. NPY is a potent angiogenic growth factor that induces angiogenesis by stimulating endothelial cell adhesion to matrix, migration, proliferation, and differentiation into capillary-like tubes (31). NPY operates with leptin in the regulation of food intake and energy expenditure (32); however, the potential for a similar relationship in angiogenesis
JOE — Volume 35, Number 6, June 2009
had not been explored. The results of the current study suggest that NPY can indeed regulate leptin production by pulp fibroblasts. NPY also had a substantial effect on ANG-2 expression in the dental pulp. ANG-2 facilitates angiogenesis in concert with VEGF, but it can lead to vessel regression in the absence of VEGF (20). Endothelial cells in contact with smooth muscle cells require ANG-2 to enable them to respond to VEGF stimulation in a cellular model of sprouting angiogenesis (33). Because the angiogenic effects of NPY have been reported to be
Regulation of Angiogenic Growth Factor Expression by Neuropeptides
831
Basic Research—Biology B
6000
7000
Concentration of VEGF (pg/ml)
Concentration of Angiogenin (pg/ml)
A 6000 5000 4000 3000 2000 1000 0
24
5000 4000 3000 2000 1000 0
48
24
Hours
C
D 600
Concentration of Leptin (pg/ml)
100
Concentration of Ang-2 (pg/ml)
48
Hours
90 80 70 60 50 40 30 20 10 0
24
500 400 300 200 100 0
48
24
Hours
48
Hours
E Concentration of bFGF (pg/ml)
30 Control 25
>50% < 50%
20
NPY SP
15
VIP CGRP
10 5 0
24
48
Hours
Figure 2. Pulp fibroblast angiogenic growth factor expression unaltered or variably altered by neuropeptides: (A) angiogenin, (B) VEGF, (C) ANG-2, (D) leptin, (E) bFGF.
mediated at least in part by VEGF (34), it is possible that ANG-2 up-regulation is a crucial step in the NPY-induced pulpal angiogenic response. EGF and PDGF-BB were produced by pulp fibroblasts in low concentration compared with other growth factors. EGF levels in tissues (35) and human dentin (4) are generally low, similar to those reported in the current study. Despite its low level, EGF might act synergistically with other angiogenic growth factors in the same way that it has been shown to synergize with TGF-b (36). The present investigation reported 832
El karim et al.
for the first time expression of PDGF-BB by pulp fibroblasts, but at lower concentration than that reported for PDGF-AB in pulp fibroblasts in vitro (14) or dentin (4). This concentration difference is not unexpected because variable tissue distribution and functional effects of PDGF dimers have been reported (37). In conclusion, this study demonstrated for the first time that angiogenin, leptin, ANG-2, and HB-EGF are synthesized and secreted by pulp fibroblasts in vitro. Furthermore, neuropeptides influence the release JOE — Volume 35, Number 6, June 2009
Basic Research—Biology of angiogenic factors by pulp fibroblasts. These findings confirm the importance of pulp fibroblasts in angiogenesis (38), and the novel role described for neuropeptides in this process opens up new possibilities for therapeutic strategies in vivo on the basis of targeting neuropeptide levels and/or neuropeptide receptors.
Acknowledgments The authors gratefully acknowledge the financial support of a TC White Research Grant from the Royal College of Physicians and Surgeons of Glasgow.
References 1. Baume LJ. The biology of pulp and dentine: a historic, terminologic-taxonomic, histologic-biochemical, embryonic and clinical survey. Monogr Oral Sci 1980;8: 1–220. 2. Guven G, Altun C, Gunhan O, et al. Co-expression of cyclooxygenase-2 and vascular endothelial growth factor in inflamed human pulp: an immunohistochemical study. J Endod 2007;33:18–20. 3. Grando ML, Westphalen BL, de Figueiredo JA, Nor JE, de Araujo FB, Fossati AC. Vascular endothelial growth factor and its relationship with the dental pulp. J Endod 2007;33:524–30. 4. Roberts-Clark DJ, Smith AJ. Angiogenic growth factors in human dentine matrix. Arch Oral Biol 2000;45:1013–6. 5. Lundy FT, Linden GJ. Neuropeptides and neurogenic inflammation in oral and periodontal inflammation. Crit Rev Oral Biol Med 2004;15:82–98. 6. Caviedes-Bucheli J, Munoz HR, Azuero-Holguin MM, Ulate E. Neuropeptides in dental pulp: the silent protagonists. J Endod 2008;34:773–88. 7. Awawdeh L, Lundy FT, Shaw C, Lamey PJ, Linden GJ, Kennedy JG. Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth. Int Endod J 2002;35:30–6. 8. El Karim IA, Lamey PJ, Ardill J, Linden GJ, Lundy FT. Vasoactive intestinal polypeptide (VIP) and VPAC1 receptor in adult human dental pulp in relation to caries. Arch Oral Biol 2006;51:849–55. 9. El Karim IA, Lamey PJ, Linden GJ, Awawdeh LA, Lundy FT. Caries-induced changes in the expression of pulpal neuropeptide Y. Eur J Oral Sci 2006;114:133–7. 10. Hokfelt T, Bartfai T, Bloom F. Neuropeptides: opportunities for drug discovery. Lancet Neurol 2003;2:463–72. 11. Tancharoen S, Sarker KP, Imamura T. et al. Neuropeptide release from dental pulp cells by RgpB via proteinase-activated receptor-2 signalling. J Immunol 2005;174: 5796–804. 12. Killough SA, Lundy FT, Irwin CR. Substance P expression by human dental pulp fibroblasts: a potential role in neurogenic inflammation. J Endod 2009;35:73–7. 13. Juarranz Y, Gutierrez-Canas I, Santiago B, Carrion M, Pablos JL, Gomariz RP. Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts. Arthritis Rheum 2008; 58:1086–95. 14. Tran-Hung L, Laurent P, Camps J, About I. Quantification of angiogenic growth factors released by human dental cells after injury. Arch Oral Biol 2008;53:9–13. 15. Yamaguchi M, Kojima T, Kanekawa M, Aihara N, Nogimura A, Kasai K. Neuropeptides stimulate production of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in human dental pulp cells. Inflamm Res 2004;53:199–204. 16. Patel T, Park SH, Lin LM, Chiappelli F, Huang GT. Substance P induces interleukin-8 secretion from human dental pulp cells. Oral Surg Oral Med Oral Pathol 2003;96: 478–85.
JOE — Volume 35, Number 6, June 2009
17. Derringer KA, Linden RW. Angiogenic growth factors released in human dental pulp following orthodontic force. Arch Oral Biol 2003;48:285–91. 18. Ohnishi T, Suwa M, Oyama T, Arakaki N, Torii M, Daikuhara Y. Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation. J Dent Res 2000;79:748–55. 19. Fett JW, Strydom DJ, Lobb RR, et al. Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 1985;24:5480–6. 20. Lobov IB, Brooks PC, Lang RA. Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci U S A 2002;99:11205–10. 21. Hu GF, Chang SI, Riordan JF, Vallee BL. An angiogenin-binding protein from endothelial cells. Proc Natl Acad Sci U S A 1991;88:2227–31. 22. Ongusaha PP, Kwak JC, Zwible AJ, et al. HB-EGF is a potent inducer of tumor growth and angiogenesis. Cancer Res 2004;64:5283–90. 23. Sierra-Honigmann MR, Nath AK, Murakami C, et al. Biological action of leptin as an angiogenic factor. Science 1998;281:1683–6. 24. Bouloumie A, Drexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res 1998;83:1059–66. 25. Salo P, Bray R, Seerattan R, Reno C, McDougall J, Hart DA. Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament. Regul Pept 2007;142: 1–6. 26. Byers MR, Taylor PE, Khayat BG, Kimberly CL. Effects of injury and inflammation on pulpal and periapical nerves. J Endod 1990;16:78–84. 27. Schroder U. Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation, and differentiation. J Dent Res 1985;64:541–8. 28. Zhang M, Fukuyama H. CGRP immunohistochemistry in wound healing and dentin bridge formation following rat molar pulpotomy. Histochem Cell Biol 1999;112: 325–33. 29. Ziche M, Morbidelli L, Pacini M, Geppetti P, Alessandri G, Maggi CA. Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells. Microvasc Res 1990;40:264–78. 30. Seegers HC, Hood VC, Kidd BL, Cruwys SC, Walsh DA. Enhancement of angiogenesis by endogenous substance P release and neurokinin-1 receptors during neurogenic inflammation. J Pharmacol Exp Ther 2003;306:8–12. 31. Zukowska Z, Grant DS, Lee EW. Neuropeptide Y: a novel mechanism for ischemic angiogenesis. Trends Cardiovasc Med 2003;13:86–92. 32. Kokot F, Ficek R. Effects of neuropeptide Y on appetite. Miner Electrolyte Metab 1999;25:303–5. 33. Korff T, Kimmina S, Martiny-Baron G, Augustin HG. Blood vessel maturation in a 3-dimensional spheroidal co-culture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J 2001;15:447–57. 34. Lee EW, Michalkiewicz M, Kitlinska J, et al. Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles. J Clin Invest 2003;111: 1853–62. 35. Carpenter G, Wahl M. The epidermal growth factor family. In: Sporn MB, Roberts AB, eds. Peptide growth factors and their receptors. New York: SpringerVerlag, 1990:69–75. 36. Thesleff I, Ekblom P, Keski-Oja J. Inhibition of morphogenesis and stimulation of vascular proliferation in embryonic tooth cultures by a sarcoma growth factor preparation. Cancer Res 1983;43:5902–9. 37. Yokose S, Kadokura H, Tajima N, et al. Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells. Cell Tissue Res 2004;315:375–84. 38. Tran-Hung L, Mathieu S, About I. Role of human pulp fibroblasts in angiogenesis. J Dent Res 2006;85:819–23.
Regulation of Angiogenic Growth Factor Expression by Neuropeptides
833
Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Ikhlas A. El karim, BDS, PhD, Gerard J. Linden, BDS, BSc, PhD, Chris R. Irwin, BSc, BDS, PhD, and Fionnuala T. Lundy, BSc, PhD Abstract Introduction: Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro. Methods: An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts. Results: Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro. Conclusions: The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair. (J Endod 2009;35:829–833)
Key Words Angiogenesis, dental pulp, growth factors, neuropeptides
From the Department of Restorative Dentistry, School of Dentistry, Royal Group of Hospitals, Belfast, United Kingdom. Address requests for reprints to Dr Ikhlas A. El karim, Department of Restorative Dentistry, School of Dentistry, Royal Group of Hospitals, Grosvenor Rd, Belfast BT12 6BP, United Kingdom. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2009 American Association of Endodontists. doi:10.1016/j.joen.2009.03.005
A
ngiogenesis is a complex multistep process of new blood vessel formation that is regulated by numerous growth factors. In the dental pulp angiogenesis is crucial for tooth development and a prerequisite for successful repair after injury and inflammation (1). Angiogenic growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF-2), and transforming growth factor (TGF-b) have been identified in human dental pulp and dentin matrix (2–4). The dental pulp is a highly innervated tissue, and pulpal nerves, through the release of neuropeptides, have been shown to play an important role in inflammation (5). Neuropeptides such as substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) are up-regulated in dental pulp from carious teeth, suggesting a role for these neuropeptides in pulpal inflammation and healing (6–9). Neuropeptides have also been implicated in mediating angiogenesis (10), and given that fibroblasts express neuropeptides (11, 12) and neuropeptide receptors (12, 13), it is possible that neuropeptides might regulate angiogenic growth factor expression by pulpal fibroblasts. Indeed, mechanical injury to pulpal fibroblasts has been shown to be associated with angiogenic growth factor release (14). The aims of this study were therefore to establish the levels of expression of a range of angiogenic growth factors by pulp fibroblasts in vitro and to determine the effects of SP, CGRP, NPY, and VIP on expression levels after 24 and 48 hours in culture.
Materials and Methods Cell Culture Human pulp fibroblasts were derived from extracted healthy molar teeth collected with consent under the approval of the Office for Research Ethics Committees Northern Ireland as previously described (12). Fibroblasts were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere of 5% CO2, 95% air. On reaching confluence, fibroblast populations were propagated at a 1:3 split ratio. Cells between passages 4 and 5 were used in this study. Cell Treatment with Neuropeptides Pulp fibroblasts were seeded in 6-well plates at a density of 3 105 cells/mL and grown to confluence. Cells were rendered quiescent by culturing in DMEM supplemented with 1% FCS for 24 hours before stimulation with neuropeptides. A concentration of 1 10–7 mmol/L (15, 16) of NPY, VIP, CGRP, and SP (Bachem, Weil am Rhein, Germany) was added to the cells (in 1% culture medium) and incubated at 37 C for either 24 or 48 hours. All studies were undertaken in triplicate, and the supernatants from each culture condition were pooled and stored at –80 C until array analysis. Pulp fibroblasts in 1% culture medium (without neuropeptide treatment) were used as controls. Angiogenic Array Quantification of angiogenic cytokines produced by pulpal fibroblasts was performed by using the Quantibody Human Angiogenesis Array (RayBiotech, Inc, Norcross, GA) to determine the concentration of 10 cytokines simultaneously: angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), bFGF, heparin-binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), leptin, platelet-derived
JOE — Volume 35, Number 6, June 2009
Regulation of Angiogenic Growth Factor Expression by Neuropeptides
829
Basic Research—Biology growth factor (PDGF-BB), placental growth factor (PIGF), and vascular endothelial growth factor (VEGF). Each cytokine was arrayed in quadruplicate, together with positive and negative controls. Cytokine standards at predetermined concentrations were used to generate a 5-point standard curve. Before addition of standard cytokines or sample, the glass chip was blocked by incubation with 100-mL sample diluent for 30 minutes. The cytokine standards or pulp fibroblast supernatant (100 ml) were added and incubated at room temperature for 1 hour. After a washing step, 80 mL of detection antibody was added (1 hour at room temp) followed by a further washing step before addition of 80 mL of Alexa Fluor 555–conjugated streptavidin for 1 hour. Fluorescent signals were visualized with a laser scanner (Axon GenePix; Molecular Devices, Sunnyvale, CA) set at 555-nm excitation, 565-nm emission, and 10-mm resolution. Data were extracted and analyzed with RayBio Q Analyzer software (RayBiotech, Inc).
Results Angiogenic Growth Factor Expression by Pulp Fibroblasts In Vitro The human angiogenic growth factors angiogenin, ANG-2, EGF, bFGF, HB-EGF, HGF, leptin, PDGF-BB, PIGF, and VEGF were detected in pulp fibroblast supernatant from control cultures at both 24 and 48 hours (Figs. 1 and 2). After 24 hours, VEGF (2923 pg/mL) and angiogenin (3626 pg/mL) were expressed at high concentrations in pulp fibroblast supernatants (>1000 pg/mL), whereas EGF (0.4 pg/mL) and PDGF-BB (3.1 pg/mL) were much less abundant (<5 pg/mL). Several angiogenic factors tended to increase after 48 hours in control cultures (angiogenin increased from 3626 to 4263 pg/mL; HGF increased from 843 to 1396 pg/mL; PIGF increased from 63 to 194 pg/mL; VEGF increased from 2923 to 3744 pg/mL), whereas others remained unchanged (EGF) or were considerably less abundant at 48 hours compared with 24 hours (ANG-2 decreased from 61 to 15 pg/mL; leptin decreased from 371 to 69 pg/mL). Angiogenic Growth Factors Substantially Increased or Decreased by Neuropeptides A neuropeptide was considered to have induced a substantial change in growth factor expression if the increase was 50% greater than the control value, or if the decrease was 50% less than the control value. The effects of individual neuropeptides on growth factor expression fell into 1 of 3 groups: (1) substantial change (either greater than or less than 50% of control values) in growth factor expression (Fig. 1), (2) no change in growth factor expression (Fig. 2), or (3) variable effects on growth factor expression (Fig. 2). CGRP and VIP induced substantial increases in the expression of PIGF after 24 hours, which dropped below control values at 48 hours (Fig. 1A). CGRP had similar effects on HGF, with levels increased above 50% of control values at 24 hours but dropping below control values at 48 hours (Fig. 1B). All the neuropeptides tested had similar inhibitory effects on HBEGF after 24 and 48 hours of treatment (Fig. 1C). VIP and SP substantially decreased EGF levels after 48 hours of treatment, whereas NPY and CGRP effects were within 50% of control values (Fig. 1D). NPY was the only neuropeptide to produce inhibitory effects on PDGF-BB at both 24 and 48 hours, whereas the other neuropeptides produced no substantial effects (Fig. 1E). Angiogenic Growth Factors Not Altered by Neuropeptides Of the 4 neuropeptides studied, none altered the levels of angiogenin or VEGF beyond greater than or less than 50% of control values (Fig. 2A, B). 830
El karim et al.
Angiogenic Growth Factors Altered to Various Extents by Neuropeptides All 4 neuropeptides had variable effects on bFGF, ANG-2, and leptin. NPY had inhibitory effects on ANG-2 and leptin at 24 hours, but levels were substantially increased to higher than control values after 48 hours of treatment (Fig. 2C, D). A similar effect was observed with bFGF, with levels substantially lower than control at 24 hours but increased to just above control after 48 hours of NPY treatment (Fig. 2E). Although SP decreased leptin levels just below 50% of control values at 24 hours, leptin levels were increased just above 50% of control values at 48 hours (Fig. 2D). Similarly, CGRP increased leptin levels just above 50% of control values at 48 hours (Fig. 2D). CGRP induced a substantial increase in the expression of bFGF at both 24 and 48 hours. However, NPY and VIP decreased bFGF levels substantially at 24 hours, with levels returning to just above control values at 48 hours (Fig. 2E).
Discussion The dental pulp is highly vascularized connective tissue with good healing potential after injury and inflammation. In part, the healing potential is related to the ability of pulp cells to secrete growth factors that are essential for cell differentiation and neovascularization. The present investigation demonstrated for the first time that angiogenin, leptin, ANG-2, PDGF-BB, and HB-EGF are synthesized and secreted by pulp fibroblasts in vitro. Angiogenin was the most abundant growth factor secreted by pulp fibroblasts; its level exceeded that of VEGF. Furthermore, the levels of all 4 newly identified angiogenic factors in the dental pulp were higher than bFGF levels, suggesting potential physiologic roles in the dental pulp. The expression of VEGF, bFGF, PIGF, EGF, and HGF by pulp fibroblasts in vitro was confirmed, supporting previous work (4, 17, 18). The novel pulp fibroblast-derived molecules (angiogenin, ANG-2, HB-EGF, leptin) identified in the current study are potent angiogenic growth factors. Angiogenin and ANG-2 have essential roles in endothelial cell proliferation (19, 20). In the presence of VEGF, ANG-2 promotes proliferation and migration of endothelial cells and stimulates sprouting of new blood vessels. Angiogenin is crucial for bFGF-stimulated and VEGF-stimulated angiogenesis (21). HB-EGF is a potent inducer of tumor growth and angiogenesis (22), and leptin has proangiogenic effects, including induction of neovascularization and formation of capillary-like structures (23, 24). This study also demonstrated for the first time the involvement of neuropeptides in regulating angiogenic growth factor expression by pulp fibroblasts. Key angiogenic factors such as HGF and PIGF were up-regulated after 24-hour exposure to neuropeptides, whereas other factors (HB-EGF, PDGF-BB, and EGF) were decreased, and some remained unchanged (angiogenin, VEGF). These findings are in agreement with previous reports (25) and suggest a complex role for neuropeptides in regulating angiogenesis and healing. The up-regulation of neuropeptides after pulpal injury and inflammation has been well-established (7, 8, 26). During pulpal repair an angiogenic response is essential for healing and reparative dentin formation (27). A role for CGRP in pulp healing and dentin bridge formation after rat molar pulpotomy has been previously suggested (28). Our results suggest that CGRP might have a role in the early stages of angiogenesis, as evidenced by the induction of HGF, PIGF, and bFGF by pulp fibroblasts after 24 hours of treatment. SP has previously been shown to enhance endothelial cell proliferation and angiogenesis in vivo (29, 30). Although SP increased EGF expression (and to lesser extent bFGF) at 24 hours, it had a tendency to inhibit expression of many of the other angiogenic factors, suggesting a degree of complexity in the
JOE — Volume 35, Number 6, June 2009
A 350
B
Concentration of PIGF (pg/ml)
Concentration of HGF (pg/ml)
Basic Research—Biology
300 250 200 150 100 50
2500
2000
1500
1000
500
0
0 24
48
24
C
D 25
0.7
Concentration of EGF (pg/ml)
Concentration of HB-EGF (pg/ml)
48
Hours
Hours
20
15
10
5
0
24
0.6 0.5 0.4 0.3 0.2 0.1 0
48
24
Hours
48
Hours
Concentration of PDGF-BB (pg/ml)
E
5 Control
4.5
>50%
4
< 50%
3.5
NPY 3
SP
2.5
VIP
2
CGRP
1.5 1 0.5 0 24
48
Hours
Figure 1. Pulp fibroblast angiogenic growth factor expression substantially altered by neuropeptides: (A) PIGF, (B) HGF, (C) HB-EGF, (D) EGF, (E) PDGF-BB.
role of SP in angiogenesis. VIP also had variable effects, inhibiting HBEGF and PDGF-BB at 24 hours but increasing PIGF at the same time point. NPY is a potent angiogenic growth factor that induces angiogenesis by stimulating endothelial cell adhesion to matrix, migration, proliferation, and differentiation into capillary-like tubes (31). NPY operates with leptin in the regulation of food intake and energy expenditure (32); however, the potential for a similar relationship in angiogenesis
JOE — Volume 35, Number 6, June 2009
had not been explored. The results of the current study suggest that NPY can indeed regulate leptin production by pulp fibroblasts. NPY also had a substantial effect on ANG-2 expression in the dental pulp. ANG-2 facilitates angiogenesis in concert with VEGF, but it can lead to vessel regression in the absence of VEGF (20). Endothelial cells in contact with smooth muscle cells require ANG-2 to enable them to respond to VEGF stimulation in a cellular model of sprouting angiogenesis (33). Because the angiogenic effects of NPY have been reported to be
Regulation of Angiogenic Growth Factor Expression by Neuropeptides
831
Basic Research—Biology B
6000
7000
Concentration of VEGF (pg/ml)
Concentration of Angiogenin (pg/ml)
A 6000 5000 4000 3000 2000 1000 0
24
5000 4000 3000 2000 1000 0
48
24
Hours
C
D 600
Concentration of Leptin (pg/ml)
100
Concentration of Ang-2 (pg/ml)
48
Hours
90 80 70 60 50 40 30 20 10 0
24
500 400 300 200 100 0
48
24
Hours
48
Hours
E Concentration of bFGF (pg/ml)
30 Control 25
>50% < 50%
20
NPY SP
15
VIP CGRP
10 5 0
24
48
Hours
Figure 2. Pulp fibroblast angiogenic growth factor expression unaltered or variably altered by neuropeptides: (A) angiogenin, (B) VEGF, (C) ANG-2, (D) leptin, (E) bFGF.
mediated at least in part by VEGF (34), it is possible that ANG-2 up-regulation is a crucial step in the NPY-induced pulpal angiogenic response. EGF and PDGF-BB were produced by pulp fibroblasts in low concentration compared with other growth factors. EGF levels in tissues (35) and human dentin (4) are generally low, similar to those reported in the current study. Despite its low level, EGF might act synergistically with other angiogenic growth factors in the same way that it has been shown to synergize with TGF-b (36). The present investigation reported 832
El karim et al.
for the first time expression of PDGF-BB by pulp fibroblasts, but at lower concentration than that reported for PDGF-AB in pulp fibroblasts in vitro (14) or dentin (4). This concentration difference is not unexpected because variable tissue distribution and functional effects of PDGF dimers have been reported (37). In conclusion, this study demonstrated for the first time that angiogenin, leptin, ANG-2, and HB-EGF are synthesized and secreted by pulp fibroblasts in vitro. Furthermore, neuropeptides influence the release JOE — Volume 35, Number 6, June 2009
Basic Research—Biology of angiogenic factors by pulp fibroblasts. These findings confirm the importance of pulp fibroblasts in angiogenesis (38), and the novel role described for neuropeptides in this process opens up new possibilities for therapeutic strategies in vivo on the basis of targeting neuropeptide levels and/or neuropeptide receptors.
Acknowledgments The authors gratefully acknowledge the financial support of a TC White Research Grant from the Royal College of Physicians and Surgeons of Glasgow.
References 1. Baume LJ. The biology of pulp and dentine: a historic, terminologic-taxonomic, histologic-biochemical, embryonic and clinical survey. Monogr Oral Sci 1980;8: 1–220. 2. Guven G, Altun C, Gunhan O, et al. Co-expression of cyclooxygenase-2 and vascular endothelial growth factor in inflamed human pulp: an immunohistochemical study. J Endod 2007;33:18–20. 3. Grando ML, Westphalen BL, de Figueiredo JA, Nor JE, de Araujo FB, Fossati AC. Vascular endothelial growth factor and its relationship with the dental pulp. J Endod 2007;33:524–30. 4. Roberts-Clark DJ, Smith AJ. Angiogenic growth factors in human dentine matrix. Arch Oral Biol 2000;45:1013–6. 5. Lundy FT, Linden GJ. Neuropeptides and neurogenic inflammation in oral and periodontal inflammation. Crit Rev Oral Biol Med 2004;15:82–98. 6. Caviedes-Bucheli J, Munoz HR, Azuero-Holguin MM, Ulate E. Neuropeptides in dental pulp: the silent protagonists. J Endod 2008;34:773–88. 7. Awawdeh L, Lundy FT, Shaw C, Lamey PJ, Linden GJ, Kennedy JG. Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth. Int Endod J 2002;35:30–6. 8. El Karim IA, Lamey PJ, Ardill J, Linden GJ, Lundy FT. Vasoactive intestinal polypeptide (VIP) and VPAC1 receptor in adult human dental pulp in relation to caries. Arch Oral Biol 2006;51:849–55. 9. El Karim IA, Lamey PJ, Linden GJ, Awawdeh LA, Lundy FT. Caries-induced changes in the expression of pulpal neuropeptide Y. Eur J Oral Sci 2006;114:133–7. 10. Hokfelt T, Bartfai T, Bloom F. Neuropeptides: opportunities for drug discovery. Lancet Neurol 2003;2:463–72. 11. Tancharoen S, Sarker KP, Imamura T. et al. Neuropeptide release from dental pulp cells by RgpB via proteinase-activated receptor-2 signalling. J Immunol 2005;174: 5796–804. 12. Killough SA, Lundy FT, Irwin CR. Substance P expression by human dental pulp fibroblasts: a potential role in neurogenic inflammation. J Endod 2009;35:73–7. 13. Juarranz Y, Gutierrez-Canas I, Santiago B, Carrion M, Pablos JL, Gomariz RP. Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts. Arthritis Rheum 2008; 58:1086–95. 14. Tran-Hung L, Laurent P, Camps J, About I. Quantification of angiogenic growth factors released by human dental cells after injury. Arch Oral Biol 2008;53:9–13. 15. Yamaguchi M, Kojima T, Kanekawa M, Aihara N, Nogimura A, Kasai K. Neuropeptides stimulate production of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in human dental pulp cells. Inflamm Res 2004;53:199–204. 16. Patel T, Park SH, Lin LM, Chiappelli F, Huang GT. Substance P induces interleukin-8 secretion from human dental pulp cells. Oral Surg Oral Med Oral Pathol 2003;96: 478–85.
JOE — Volume 35, Number 6, June 2009
17. Derringer KA, Linden RW. Angiogenic growth factors released in human dental pulp following orthodontic force. Arch Oral Biol 2003;48:285–91. 18. Ohnishi T, Suwa M, Oyama T, Arakaki N, Torii M, Daikuhara Y. Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation. J Dent Res 2000;79:748–55. 19. Fett JW, Strydom DJ, Lobb RR, et al. Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 1985;24:5480–6. 20. Lobov IB, Brooks PC, Lang RA. Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci U S A 2002;99:11205–10. 21. Hu GF, Chang SI, Riordan JF, Vallee BL. An angiogenin-binding protein from endothelial cells. Proc Natl Acad Sci U S A 1991;88:2227–31. 22. Ongusaha PP, Kwak JC, Zwible AJ, et al. HB-EGF is a potent inducer of tumor growth and angiogenesis. Cancer Res 2004;64:5283–90. 23. Sierra-Honigmann MR, Nath AK, Murakami C, et al. Biological action of leptin as an angiogenic factor. Science 1998;281:1683–6. 24. Bouloumie A, Drexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res 1998;83:1059–66. 25. Salo P, Bray R, Seerattan R, Reno C, McDougall J, Hart DA. Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament. Regul Pept 2007;142: 1–6. 26. Byers MR, Taylor PE, Khayat BG, Kimberly CL. Effects of injury and inflammation on pulpal and periapical nerves. J Endod 1990;16:78–84. 27. Schroder U. Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation, and differentiation. J Dent Res 1985;64:541–8. 28. Zhang M, Fukuyama H. CGRP immunohistochemistry in wound healing and dentin bridge formation following rat molar pulpotomy. Histochem Cell Biol 1999;112: 325–33. 29. Ziche M, Morbidelli L, Pacini M, Geppetti P, Alessandri G, Maggi CA. Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells. Microvasc Res 1990;40:264–78. 30. Seegers HC, Hood VC, Kidd BL, Cruwys SC, Walsh DA. Enhancement of angiogenesis by endogenous substance P release and neurokinin-1 receptors during neurogenic inflammation. J Pharmacol Exp Ther 2003;306:8–12. 31. Zukowska Z, Grant DS, Lee EW. Neuropeptide Y: a novel mechanism for ischemic angiogenesis. Trends Cardiovasc Med 2003;13:86–92. 32. Kokot F, Ficek R. Effects of neuropeptide Y on appetite. Miner Electrolyte Metab 1999;25:303–5. 33. Korff T, Kimmina S, Martiny-Baron G, Augustin HG. Blood vessel maturation in a 3-dimensional spheroidal co-culture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J 2001;15:447–57. 34. Lee EW, Michalkiewicz M, Kitlinska J, et al. Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles. J Clin Invest 2003;111: 1853–62. 35. Carpenter G, Wahl M. The epidermal growth factor family. In: Sporn MB, Roberts AB, eds. Peptide growth factors and their receptors. New York: SpringerVerlag, 1990:69–75. 36. Thesleff I, Ekblom P, Keski-Oja J. Inhibition of morphogenesis and stimulation of vascular proliferation in embryonic tooth cultures by a sarcoma growth factor preparation. Cancer Res 1983;43:5902–9. 37. Yokose S, Kadokura H, Tajima N, et al. Platelet-derived growth factor exerts disparate effects on odontoblast differentiation depending on the dimers in rat dental pulp cells. Cell Tissue Res 2004;315:375–84. 38. Tran-Hung L, Mathieu S, About I. Role of human pulp fibroblasts in angiogenesis. J Dent Res 2006;85:819–23.
Regulation of Angiogenic Growth Factor Expression by Neuropeptides
833