Glucocorticoids control bone microvascular barrier function via Connexin 43 pathway

Introduction: Glucocorticoids (GCs), such as prednisone, methylprednisolone, and dexamethasone, are potent treatments for inflammatory and autoimmune disorders. Despite their proven clinical benefit, patients under prolonged GC therapy are at high risk of developing bone-related adverse effects, such as osteoporosis. GC-induced osteoporosis, most commonly occurring before the age of 50, is the most prevalent type of secondary osteoporosis, associated with increased fracture risk and bone loss. Although the predominant effect of GCs on bone is linked with the loss of function of osteoblasts and osteoclasts, their role in bone microvasculature is incompletely understood. This study aims to elucidate pathogenic mechanisms underlying GC-induced osteoporosis by investigating the cell adhesion mechanisms that mediate bone microvascular barrier function and osteoblast-endothelial interaction in response to GCs. Endothelial and osteoblast function in bone results from several transduction cascades, including gap junction molecules. Our studies are particularly focused on connexin 43 (Cx43), which controls endothelial cell-cell contact, angiogenesis, and osteoblast/osteoclast differentiation. Deciphering the intricate interplay between glucocorticoids, Cx43, and osteoblast-endothelial cell interactions is crucial for a deeper understanding of bone pathophysiology. Unraveling these complex mechanisms may pave the way for novel preventive and therapeutic strategies to preserve bone health and improve clinical outcomes for patients susceptible to osteoporosis due to corticosteroid treatment.

Materials and

Methods: To study the role of bone microvasculature and the endothelial-osteoblast interaction in normal and disease states, we engineered a novel 3D bicellular model using a microfluidic device. This platform consists of 3D perfusable endothelium embedded within a collagen I matrix surrounded by human osteoblast (HOBs) cells. For the GCs experiments, endothelial cells (ECs) and HOBs were treated for 18 days in the presence of 200 nmol/L hydrocortisone (HYD), 100 nmol/L prednisone (PRED), and 100 nmol/L prednisolone (PREDS). Initially, we evaluated the osteogenic potential of osteoblasts under normal and GC-treated conditions by using alkaline phosphatase (ALP), alizarin red (AR), and Von kossa (VK) staining. Next, we examined the role of GCs in the microvascular barrier function by imaging the diffusion of fluorescent dextran (70 kDa Texas Red, Thermo Fisher) in real-time with a confocal microscope (LSM 800, Carl Zeiss) at 10x magnification. Analysis of the mean intensity of those images resulted in determining the diffusive permeability coefficient (Pd). In our mechanistic studies, protein analysis by immunoblotting was performed for phosphorylated and total levels of Connexin 43. For the in vivo studies, prednisolone pellets were implanted subcutaneously in the dorsal scapular region of 4-6 months-old Swiss Webster mice. 60 days post pellet implantation, the mice were euthanized, the long bones and skull were harvested, and subjected to histopathological analyses. Cx43 expression and bone microarchitecture were visualized by immunohistochemistry (IHC).

Results, Conclusions, and Discussions: Initially, our results showed that the presence of GCs decreased osteogenic function both in vivo and in vitro. Specifically, our in vitro studies demonstrated that the presence of GCs reduced HOB functionality by reducing ALP activity (11-fold), calcium deposition (performed by AR staining) by 4.5-fold, and mineral deposition (performed by VK staining) by 6.4-fold compared to untreated conditions (p < 0.05). Also, GCs led to a 5-fold decrease in the mineral deposition based on VK analysis and a 10-times decrease in the number of blood vessels. In addition, our in vivo data showed a 5-fold decrease in mineral deposition based on VK staining, indicating that GCs diminished the osteoblastic function. Next, we investigated the role of GCs in bone microvasculature. Specifically, our in vitro data showed that vascular leakiness (Pd ) increased 5-fold (HYD), 4-fold (PRED), and 7.8-fold (PREDS) compared to the untreated conditions. Using the microfluidic device setup, we demonstrated that the osteoblast-endothelial cell interface integrity was disrupted via loss of junctional Cx43 upon GC treatment. Also, blockage of Cx43 expression showed upregulation of vascular leakiness by 4.5-fold (p < 0.05), while bone tissues showed loss of Cx43 in prednisolone-treated animals, indicating that Cx43 is essential in bone microvascular function. Finally, immunoblotting analysis of ECs-HOBs co-culture lysates showed that GCs induce activation of the MAPK pathway, which in turn regulates Cx43 phosphorylation and destabilizes the cell-cell junctions.
Overall, our in vitro and in vivo results offer insights into the subcellular cascade of events that underlie osteoporosis and may open new avenues for mitigation of corticosteroid-dependent adverse effects on bone health.

Acknowledgements (Optional): This work was supported by the National Institute of Dental and Craniofacial Research (NIDCR) of the National Institutes of Health (R01DE031046). We gratefully acknowledge the Microscopy and Imaging Shared Resource (MISR) and the Lombardi Comprehensive Cancer Center grant (P30-CA051008).