Changes in mandibular CDH5, CXCL1, and PECAM1 expression following exposure to bisphosphonate and molar extraction suggest a loss of vascular endothelial cell barrier integrity plays a role in MRONJ

Abstract

Objectives: Medication related osteonecrosis of the jaw (MRONJ) is a disorder characterized by loss of blood supply to the jaws and death to the bone. In our previous work, we created a rat model of MRONJ by multiple injections of 60ug/Kg zoledronic acid (ZA) via tail vein followed by extraction of a single first molar. We have previously shown in this model a decrease in the vasculature of the jaws and a delay in bone healing after 6 weeks. The current study was designed to look at mRNA expression and immunohistochemical localization of three endothelial cell markers (CDH5, CXCL1 and PECAM1) 6 weeks following ZA injection with and without molar extraction and in saline injected rats as controls. The objective of this study is to determine if the expression of these markers may serve to identify clinically significant changes in vascular endothelium barrier function associated with the onset of MRONJ. Methods: Using RT-PCR, we analyzed the expression of mRNA for angiogenic markers, six weeks after injection with either 60ug/Kg ZA or saline as a control and in ZA injected rats following first molar extraction. Routine immunohistochemical procedures with antibodies specific for rat marker proteins were used to study the immunohistochemical localization of proteins translated from the mRNA angiogenic markers with significant expression. All tissues were processed together under identical conditions. Results: We found a decrease in the mRNA expression of CDH5, and PECAM1 and an increase in CXCL1 expression six weeks after extraction and ZA injection in our rat model of MRONJ. Rat monoclonal antibodies specific for protein epitopes expressed by these genes (VE-cadherin, C-X-C motif ligand 1(CXCl1), and PECAM-1) were then used to localize proteins for these markers in tissues from ZA, saline-injected control rats, and MRONJ rats. The results show the absence of immunohistochemical localization of PECAM-1 and VE-cadherin marker proteins (-) in tissues from the MRONJ rats but strong (++++) in tissue from the saline-injected Control rats. The results also show the localization of the immunohistochemical staining for CXCl1 was high (++++) in the MRONJ rats after six weeks but absent (-) in the Controls. No mRNA or immunohistochemical localization for the three markers was seen in the rats treated with ZA without extraction. Conclusions: Our study identifies markers associated with a loss of vascular endothelial cell barrier integrity suggesting a role for this mechanism in MRONJ. This finding further suggests that CDH5, CXCL1 and PECAM1 may be useful as early markers for the changes seen in blood vessels after bisphosphonate exposure but before the frank onset of MRONJ.