Association between UHMWPE particle-induced inflammatory osteoclastogenesis and expression of RANKL, VEGF, and Flt-1 in vivo
Introduction
Osteolysis around knee and hip implants that subsequently leads to aseptic loosening has become a major clinical problem, and occurs in association with the formation of a vascularized granulomatous tissue in response to wear debris [1]. Since neovascularization is a hallmark of both bone remodeling and inflammation, the pro-angiogenetic status in periprosthetic tissue may augment the inflammatory response to wear debris [1], [2]. Recent studies have provided insights into molecular mechanisms that may be critical to this process. Vascular endothelial growth factor (VEGF), the most potent angiogenic growth factor, is produced by many cells including macrophages, lymphocytes, and osteoblasts [3], [4]. VEGF expression is regulated by growth factors, hormones, and cytokines [4], [5], [6], [7]. The receptors for VEGF belong to the tyrosine kinase receptor family, and two specific VEGF receptors have been identified to date; VEGF receptor-1 (VEGFR-1; fms-like tyrosine kinase, Flt-1) and VEGFR-2 (Flk-1/KDR) [8]. Current findings suggest that Flt-1 may play a critical role in the process of inflammation and osteoclastogenesis, based upon the observation that (i) Flt-1 is expressed on cells of the monocyte-macrophages lineage and is involved in macrophage activation [9]; (ii) Flt-1 signaling is a driving force in the recruitment of macrophages to inflamed tissues [10], [11], [12], [13], and (iii) Flt-1 is involved in both osteoclast formation (osteoclastogenesis) [14] and osteoclast bone resorptive activity [15]. Since VEGF signaling is involved in the process of osteoclastogenesis [14], [15], [16], receptor activator of nuclear factor kappa B ligand (RANKL) and its receptor RANK [17], [18] may act as a common mediator for inflammatory osteoclastogenesis [19]. RANKL and VEGF both regulate their activity through multiple signal transduction pathways, which have not yet been completely characterized. These two cytokines are known to be present in the local bone environment, and their corresponding receptors RANK (for RANKL) and Flt-1 (for VEGF) may be detected in osteoclasts and osteoclast precursor cells [14], [20], [15]. Both factors stimulate the recruitment of osteoclasts in a similar manner with respect to kinetics and signal transduction pathway [21], [22].
We have demonstrated that ultra-high-molecular-weight (UHMWPE) particles initiate a significantly higher RANK/RANKL gene expression in a murine model of tissue inflammation and osteoclastogenesis [23]. However, the possible interrelationship between VEGF and RANKL biological signaling during inflammatory osteoclastogenesis remains unclear. Using a mouse osteolysis model [24], we have recently demonstrated that stimulation by UHMWPE particles significantly increased VEGF expression, and immunohistology revealed that intense VEGF staining occurred predominantly on inflammatory cells around UHMWPE deposits. These findings suggest that VEGF signaling may be actively involved in the process of UHMWPE particle-induced inflammatory osteolysis. The purpose of this study was to test the hypothesis that amelioration of wear debris-induced inflammatory osteoclastogenesis by VEGF inhibitor treatment occurs through the down regulation of RANK, RANKL, and VEGF receptor genes in a mouse osteolysis model.
Section snippets
UHMWPE particles
UHMWPE particles (the generous gift of Dr. John Cuckler, University of Alabama, Birmingham, AL) were used to stimulate inflammatory reactions. The size and distribution of UHMWPE particles was evaluated with a Coulter particle counter and by scanning electron microscopy as described previously [25]. The UHMWPE particles had a mean particle diameter of 2.6 μm (range from <0.6 to 21 μm). The particles were washed in 70% ethanol solution to remove endotoxin and heat sterilized, and the absence of
Expression of VEGF and VEGF receptors in mouse pouch tissues
The gene expression of VEGF and VEGF receptors in the pouch tissues were determined by both immunostaining and RT-PCR. As shown in Fig. 1A, intense VEGF staining was observed in UHMWPE particle-stimulated pouches, compared with low expression in the PBS controls. VEGF staining was predominantly observed on the inflammatory cells surrounding UHMWPE deposits. VEGF treatment exerted little effect on the level of VEGF staining, either in the presence or absence of UHMWPE stimulation. However, VEGF
Discussion
This study indicates that UHMWPE particle stimulation significantly increases VEGF gene activity, but exerts a lesser influence on the levels of Flt-1 and Flk-1 gene activity in this model. UHMWPE particle-stimulated VEGF production was markedly reduced by VEGF inhibitor treatment. Pouch membrane macrophages were the predominant cells expressing both VEGF and Flt-1, and there was a positive association between tissue inflammation status and the levels of VEGF and Flt-1 gene activity. Both RANK
Conclusion
The present study shows that VEGF is actively involved in the regulation of RANK/RANKL gene expression, and exerts a regulatory effect on the development of UHMWPE particle-induced inflammatory osteoclastogenesis. These observations warrant future investigations to elucidate the possible interrelationship between these two cytokines.
Acknowledgments
The authors acknowledge the excellent technical assistance of Ms. Lois Mayton. Striker Company has provided support for this project.
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2019, BoneCitation Excerpt :This demonstrated that VEGF and angiogenesis are necessary for bone generation and remodeling, establishing the role of VEGF as a growing area of interest. Further, it was shown that under inflammatory conditions, exposure to orthopedic particles lead to increased VEGF expression both at the mRNA and protein levels [5,28] and regulation of osteoclasts. These studies lend credence to the critical role of VEGF in homeostatic bone generation and regeneration as well as its contribution to inflammatory, particle-induced, osteolysis.