Synthetic Integrin-binding Peptides Promote Adhesion and Proliferation of Human Periodontal Ligament Cells in vitro

Abstract

Periodontal ligament (PDL) cells have been shown to express several integrins (α_v, α₅, β₁, β₃) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [³H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands for the human PDL cells, and that they could provide a basis for the development of new strategies aimed at the regeneration of the periodontium.

Keywords

RGD oligopeptides integrins collagens periodontium.

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References

Brooks PC , Montgomery AM , Rosenfeld M. , Reisfeld R. , Hu T. , Klier G. , et al. (1994). Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenetic blood vessels. Cell 79:1157-1164.

Caffesse RG , Nasjleti CE , Anderson GB , Lopatin DE , Smith BA , Morrison EC (1991). Periodontal healing following guided tissue regeneration with citric acid and fibronectin application. J Periodontol 62:21-29.

Cho MI , Garant PR , Lee YL (1988). Immunocytochemical in vivo localization of fibronectin-rich contact sites on fibroblasts of normal periodontal ligament and inflamed gingiva . J Periodont Res 23:230-238.

Choi SY , Nilveus RE , Minutello RD , Zimmerman GJ , Wikesjo UM (1993). Effect of a collagen matrix on healing in periodontal fenestration defects in dogs. J Periodontol 64:878-882.

Clark EA , Brugge JS (1995). Integrins and signal transduction pathways: the road taken. Science 268:233-239.

Furuto DK , Miller EJ (1987). Isolation and characterization of collagens and procollagens. Meth Enzymol 144:41-61.

Gottlow J. (1993). Guided Tissue Regeneration using bio-resorbable and non-resorbable devices: Initial healing and long term results. J Periodontol 64:1157-1165.

Grzesik WJ , Gehron Robey P. (1994). Bone matrix RGDglycoproteins : immunolocalization and their interaction with human primary osteoblastic bone cells in vitro . J Bone Miner Res 9:487-496.

Harden JF (1983). Periodontal disease in America: a personal and national tragedy. J Public Health Dent 43:106-117.

10.

Hynes RO (1992). Integrins, versatility, modulation, and signaling in cell adhesion. Cell 69:11-25.

11.

Iglhaut J. , Aukhil I. , Simpson DM , Johnston MC , Koch G. ( 1988). Progenitor cell kinetics during guided tissue regeneration in experimental periodontal wounds. J Periodont Res 23 :107-117.

12.

Ivanov B. , Grzesik W. , Robey FA (1995). Synthesis and use of a new bromoacetyl-derivatized heterotrifunctional amino acid for conjugation of cyclic RGD-containing peptides derived from human bone sialoprotein. Bioconjugate Chem 6:269-277.

13.

Lynch SE , Williams RC , Polson AM , Howell TH , Reddy MS , Zappa UE , et al. (1989). A combination of platelet-derived and insulin-like growth factors enhances periodontal regeneration. J Clin Periodontol 16:545-548.

14.

Mailhot JM , Schuster GS , Garnick JJ , Hanes PJ , Lapp CA , Lewis JB (1995). Human periodontal ligament and gingival fibroblast response to TGF-beta 1 stimulation. J Clin Periodontol 22:679-685.

15.

Matsuura M. , Herr Y. , Han KY , Lin WL , Genco RJ , Cho MI (1995). Immunohistochemical expression of extracellular matrix components of normal and healing periodontal tissues in the beagle dog. J Periodontol 66:579-593.

16.

Mendieta C. , Caravana C. , Fine DH ( 1990). Sorption of fibronectin to human root surfaces in vitro . J Periodontol 61:254-260.

17.

Meredith JE Jr , Winitz S. , Lewis JM , Hess S. , Ren X.-D. , Renshaw MW , et al. (1996). The regulation of growth and intracellular signaling by integrins. Endocrine Rev 17:207-220.

18.

Pierschbacher MD , Ruoslahti E. (1987). Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J Biol Chem 262 :17294-17298.

19.

Ruoslahti E. , Reed JC ( 1994). Anchorage dependence, integrins, and apoptosis. Cell 77:477-478.

20.

Rutherford RB , TrailSmith MD , Ryan ME , Charette MF (1992a). Synergistic effects of dexamethasone on platelet-derived growth factor mitogenesis in vitro. Arch Oral Biol 37 :139-145.

21.

Rutherford RB , Niekrash CE , Kennedy JE , Charette MF (1992b). Platelet-derived and insulin-like growth factors stimulate regeneration of periodontal attachment in monkeys. J Periodont Res 27:285-290.

22.

Smith BA , Smith JS , Caffesse RG , Nasjleti CE , Lopatin DE , Kowalski CJ (1987). Effect of citric acid and various concentrations of fibronectin on healing following periodontal flap surgery in dogs. J Periodontol 58:667-673.

23.

Somerman MJ , Foster RA , Imm GM , Sauk JJ , Archer SY (1989). Periodontal ligament cells and gingival fibroblasts respond differently to attachment factors in vitro. J Periodontol 60 :73-77.

24.

Somerman MJ , Sauk JJ , Foster RA , Norris K. , Dickerson K. , Argraves WS (1991). Cell attachment activity of cementum: bone sialoprotein II identified in cementum. J Periodont Res 26:10-16.

25.

Steffensen B. , Duong AH , Milam SB , Potempa CL , Winborn WB , Magnusson VL , et al. (1992). Immunohistological localization of cell adhesion proteins and integrins in the periodontium. J Periodontol 63:584-592.

26.

Terranova VP (1993). Biologically active factors in the treatment of periodontal disease. Curr Opin Periodontol 129-135.

27.

Uitto VJ , Larjava H. , Peltonen J. , Brunette DM (1992). Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells. J Dent Res 71:1203-1211.

28.

van der Pluijm G. , Vloedgraven HJ , Ivanov B. , Robey FA , Grzesik WJ , Robey PG , et al. (1996). Bone sialoprotein peptides are potent inhibitors of breast cancer cell adhesion to bone. Cancer Res 56:1948-1955.

29.

Varner JA , Brooks PC , Cheresh DA (1995). The integrin αvβ3: angiogenesis and apoptosis . Cell Adhesion Commun 3 :367-374.

30.

Wary K. , Mainiero F. , Isakoff SJ , Marcantonio EE , Giancotti FG (1996). The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell 87:733-743.

31.

Yamada KM , Miyamoto S. (1995). Integrin transmembrane signaling and cytoskeletal control. Curr Opin Cell Biol 7 :681-689.

32.

Yamauchi M. , Kuboki Y. , Sasaki S. , Mechanic GL (1986). New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament. Biochemistry 25:1997-2002.