Zoledronic Acid Disrupts VEGFR2 Intracellular Trafficking in Endothelial Cells

Oral Abstract Session 5 patients with disc displacement without reduction of the temporomandibular joint (TMJ). Methods: 120 patients with a symptomatic disc displacement without reduction that did not respond to a conservative therapy were recruited. They were randomly assigned into two groups. One group of patients received three injections of sodium hyaluronate in the superior joint space and the other one in the inferior joint space. Patient’s TMJ status and clinical symptoms were evaluated at 3 and 6 months follow-up. The clinical parameters recorded were maximal mouth opening (MMO), pain intensity on a visual analogue scale (VAS), and a modified Helkimo’s clinical dysfunction index. The results were analyzed with ANCOVA. Results: 50 patients of the superior and 54 of the joint space injection group attended the follow up appointments. MMO, VAS and Helkimo’s index improved at the 3 months and 6 months follow-up appointments in both groups. At the 3 months follow-up the improvement in TMJ pain was significantly larger in the inferior than in the superior injection group (p⬍0.001), while the improvements in MMO and TMJ function were similar. At the 6 months follow-up the improvements in MMO (p⬍0.005), VAS (p⬍0.001) and Helkimo’s index (p⬍0.001) were significantly larger in the inferior than the superior joint injection group. Conclusion: This study demonstrated that the inferior joint space injection with sodium hyaluronate is a valid method for the treatment of disc displacement without reduction and should therefore be considered as a treatment modality as it appears to give patients a better long term clinical outcome than the superior joint space injection.

Zoledronic Acid Disrupts VEGFR2 Intracellular Trafficking in Endothelial Cells David L. Basi, DMD, PhD, University of Minnesota, 515 Delaware Street SE, 7-174 Moos Tower, Minneapolis, MN 55455 (Mariash A) Statement of the Problem: Long-term use of nitrogencontaining bisphosphonates (n-BISs) are associated with jaw necrosis. Tooth extraction is the most frequent precipitating factor. Proper healing after tooth extraction requires angiogenesis, which is inhibited by n-BIS in vitro and in vivo. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal for endothelial cells. N-BIS inhibit VEGF responses in endothelial cells (2). The molecular mechanism(s) that account for these attenuated responses to VEGF are not known. VEGF receptor-2 (VEGFR2) is the main signaling receptor for VEGF in endothelial cells. Control of VEGF signaling involves VEGFR2 internalization and intracellular trafficking. Caveolae-mediated endocytosis is a recognized 66

means of VEGFR2 internalization. Caveolin-1 is the main protein component found within caveolae and mediates VEGFR2 endocytosis and VEGFR2 signal transduction. Caveolin-1-mediated VEGFR2 endocytosis within endothelial cells is not completely understood but involves guanosine triphosphate (GTPase). These GTPases are inhibited by n-BIS. We, therefore, hypothesize that n-BIS disrupts VEGFR2 trafficking in endothelial cells. Materials and Methods: We investigated the effect of zoledronic acid (ZOL), a n-BIS, on caveolin-1-mediated VEGFR2 trafficking in treated human umbilical vein endothelial cells (HUVECs) using confocal microscopy, western blotting and real-time PCR (RT-PCR). Results: HUVECs treated with 12.5␮M ZOL for 48 hrs demonstrated a statistically significant increase in the percent of Caveolin-1 colocalized with VEGFR2 (p⫽0.0001) compared to non-treated cells. In addition, the quantity of VEGFR2/cell increased with ZOL treatment (p⫽0.015). Western blotting confirmed that 48hrs of ZOL treatment significantly increased the total amount of VEGFR2 (p⫽0.004) and caveolin-1 (p⫽0.004) protein in HUVECs compared to non-treated cells. To determine if ZOL increased the quantity of VEGFR2 or caveolin-1 protein by induction of VEGFR2- or Caveolin1-specific mRNA, we incubated endothelial cells with or without 12.5␮M ZOL for 2, 4, 24 and 48hrs and then analyzed the isolated RNA for VEGFR2- and caveolin-1specific mRNA by RT-PCR. We found that the relative amount of VEGFR2- or Caveolin-1-specific mRNA was not significantly increased by 12.5␮M ZOL at the time points tested. N-BIS prevents the formation of geranylgeranyl pyrophosphate by inhibiting farnesyl pyrophosphate synthase within the mevalonate pathway (1). Geranylgeranyl pyrophosphate is required for the prenylation and subsequent membrane targeting of small GTPases. Because GTPases are involved in endocytosis and we theorize that VEGFR2 trafficking is altered by ZOL, we hypothesized that ZOL inhibition of the mevalonate pathway is involved with increased caveolin-1 and VEGFR2 colocalization. Therefore, we treated HUVEC cultures with 12.5 ␮M ZOL and/or 2 ␮M geranylgeranyl pyrophosphate for 48hrs, then assessed for VEGFR2 and caveolin-1 colocalization by confocal microscopy. The addition of geranylgeranyl pyrophosphate to ZOL treated HUVECs reduced (p⫽0.0001) the percent of caveolin-1 colocalized with VEGFR2 and the accumulation of VEGFR2 within HUVECs during ZOL treatment. Statistical Method: 2- and 1-way ANOVAs with Tukey multiple comparison adjustments. Differences were considered significant at the 0.05 level. Conclusions: The data suggests that the increased Caveolin-1/VEGFR2 interactions are due to an accumulation of VEGFR2 and Caveolin-1 within the ZOL-treated endothelial cells. Furthermore, this ZOL-induced aberrant VEGFR2 accumulation and interaction with caveoAAOMS • 2008

Oral Abstract Session 5 lin-1 is not likely due to induction of mRNA transcription, but disruption of the mevalonate pathway. References Fleisch H. Bisphosphonates: mechanisms of action. Endocr Rev 19: 80-100, 1998 Wood J, Bonjean K, Ruetz S, Bellahcene A, Devy L, Foidart JM, Castronovo V, and Green JR. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther 302: 1055-1061, 2002

Effect of Nitric Oxide Donor on IL-6 and PDGF-BB: Comparison of Diabetic Versus Normal Tissue Under Hypoxic and Normoxic Conditions Andrew P. Wightman, DMD, Wilford Hall Medical Center, Lackland Air Force Base, Department of Oral and Maxillofacial Surgery, 59th Medical Wing, 59th Dental Squadron, 220 Bergquist Dr. STE 1, San Antonio, TX 78236 (Myers AD; Childress RW; Dean DD; Sylvia VL) Statement of the Problem: CDC estimates that 20.8 million people in the US have diabetes. Vascular changes in diabetic tissues result in decreases in oxygenation, platelet derived growth factor (PDGF), and nitric oxide (NO). Fibroblasts and collagen production are significantly reduced in cells from diabetic patients1. Decreased NO production and increased matrix metalloproteinase 8 and 9 occur in wounds of diabetic patients2. Diabetic wounds demonstrate increased inflammatory cytokine secretion, including Interleukin-6. This lab has also demonstrated that the addition of an NO donor can reduce the MMP-8 and MMP-9 expression by fibroblastic cells3. Materials and Methods: Human skin fibroblasts from a diabetic subject were compared to fibroblasts from a non-diabetic patient grown under normoxic and hypoxic conditions. IL-6 and PDGF production were measured. Fibroblasts from a 31yo diabetic and 32yo non-diabetic patients were obtained from the Coriell Institute. Fibroblasts were cultured to confluence and treated with 1nM, 10nM, or 100nM of long-acting SNitrosoglutathione(SNOG) or short-acting FK409 (NOR3) NO donor for 1, 3, or 7 days. Cells were cultured under normoxic (20% oxygen) or hypoxic (2% oxygen) conditions. Appropriate untreated controls were included in all experiments. At harvest, conditioned media from the cultures were assayed for production of IL-6 or PDGF-BB. IL-6 was measured using a ChemiKine® Human Interleukin-6 Sandwich ELISA kit (Chemicon International). PDGF-BB was measured using a Quantikine® Human Immunoassay (R&D Systems). Method of Data Analysis: The data were collected and the mean ⫾ standard error of the mean (SEM) was calculated for each treatment. The data were analyzed by AAOMS • 2008

ANOVA; where significant differences were found, a two-tailed t-test was performed. P-values less than 0.05 were considered significant. Results: After 1, 3, and 7 days in culture, normal fibroblasts produced 23% as much IL-6 as diabetic cells under normoxic conditions. Under hypoxic conditions, normal cells produced an even smaller relative amount of IL-6 (14%). The production of IL-6 by diabetic, but not normal, fibroblasts increased with time in culture. Under normoxic conditions, IL-6 production by diabetic fibroblasts increased from 4.34ng/mg protein to 6.94ng/mg protein from day 1 to day 7, under hypoxic conditions, IL-6 increased from 11.23ng/mg protein to 17.98ng/mg protein. NOR3 had no effect on IL-6 production by any cells under either condition. Treatment with SNOG, decreased IL-6 production by both normal and diabetic fibroblasts, with greater effect on diabetic fibroblasts. Under normoxic conditions, IL-6 production by diabetic cells was dosedependently decreased by up to 81% at the 100nM dose. A smaller response was observed with normal fibroblasts. Only the 100nM dose resulted in a significant decrease in IL-6 production at all three time points. Cells cultured under hypoxic conditions had similar results, but the magnitude was greater in diabetic cells. PDGF-BB was produced by both normal and diabetic fibroblasts. Normal fibroblasts under normoxic conditions synthesized twice as much PDGF-BB as diabetic cells. Under hypoxic conditions, normal cells produced five times the amount of PDGF synthesized by diabetic cells. There were no significant changes in PDGF-BB production with NO donor treatment. Conclusions: Production of PDGF-BB, is significantly reduced under hypoxic conditions in both normal and diabetic cells, but the effect is much greater in the diabetic cells. Synthesis of PDGF-BB by diabetic cells is more influenced by hypoxia than normal cells. Treatment with the NO donor compounds had no effect on PDGF-BB production. Production of IL-6, an inflammatory mediator, is increased in diabetic cells under both normoxic and hypoxic conditions. The addition of SNOG, significantly decreased the amount of IL-6 produced by normal and diabetic cells, but the effect was greater in the diabetic cells. References Seppala, B., Sorsa, T., Ainamo, J., Morphometric analysis of cellular and vascular changes in gingival connective tissue in long-term insulindependent diabetes. Journal of Periodontology. 68(12):1237-45, 1997 Dec Lobmann, R., Ambrosch, A., Schultz, G., Waldmann, K., Schieweck, S., Lehnert H. Expression of matrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients. Diabetologia, 45:1011-101, 2002 Burrow JW, Koch JA, Chuang H-H, Zhong W, Dean DD, and Sylvia VL: Nitric Oxide Donors Selectively Reduce the Expression of Matrix Metalloproteinases-8 and -9 by Human Diabetic Skin Fibroblasts. Journal of Surgical Research, 140:90-98, 2007

67