Nitric Oxide Controls VE-Cadherin-Mediated Vascular Integrity in Periapical Granulomas

Objectives: In order to determine the role of nitric oxide in controlling vascular endothelial (VE-) cadherin-mediated vascular integrity in chronic inflammation, surgically removed periapical granulomas and cultured human umbilical vein endothelial cells (HUVECs) were examined.

Methods: Periapical lesions (n=25) were removed at the time of endodontic surgical treatment and were cut into two portions. One portion was used for the preparation of paraffin sections and stained with hematoxylin and eosin. Another portion was fixed with 4% paraformaldehide in PBS and was then frozen in OCT compound. The specimens pathologically diagnosed as periapical granulomas (n=20) were used for the following studies, and radicular cysts (n=5) were excluded from this experiment. The expression of inducible NO synthase (iNOS) and VE-cadherin in periapical granulomas were examined using two-color immunofluorescence microscopy and real-time PCR. HUVECs were stimulated with IFNg, TNFa, IL-1b and LPS extracted from Porphyromonas gingivalis for 20 h, and quantitative analyses of iNOS and VE-cadherin protein and mRNA expression were performed using ELISA and real-time PCR, respectively.

Results: Protein and mRNA expression levels for iNOS and VE-cadherin in periapical granulomas varied from patient to patient, but that more iNOS than VE-cadherin was expressed. Stimulation of the cultured HUVECs induced the expression of iNOS protein (13.8±0.8 ng/ml), as compared with non-stimulated controls (3.2±0.6 ng/ml). On the other hand, it reduced the expression of VE-cadherin protein (0.4±0.1 ng/ml), as compared with non-stimulated controls (1.3±0.1 ng/ml, respectively). The expression of iNOS mRNA in cultured HUVECs increased, whereas that of VE-cadherin decreased, as compared with non-stimulated controls. The protein and mRNA levels of iNOS and VE-cadherin had opposite expression profiles in HUVECs.

Conclusion: These data are consistent with a hypothesis suggesting that NO could control cell-cell junctions by promoting the localized removal of VE-cadherin from endothelial cell junctions in chronic inflammation.