Chantal Séguin

Glucocorticoids in osteonecrosis of the femoral head: A new understanding of the mechanisms of action

Abstract Glucocorticoid (GC) usage is the most common non-traumatic cause of osteonecrosis of the femoral head (ON). Despite the strong association of GC with ON, the underlying mechanisms have been unclear. Investigators have proposed both direct and indirect effects of GC on cells. Indirect and direct mechanisms remain intimately related and often result in positive feedback loops to potentiate the disease processes. However, the direct effects, in particular apoptosis, have recently been shown to be increasingly important. Suppression of osteoblast and osteoclast precursor production, increased apoptosis of osteoblasts and osteocytes, prolongation of the lifespan of osteoclasts and apoptosis of endothelial cells (EC) are all direct effects of GC usage. Elevated blood pressure through several pathways may raise the risk of clot formation. High-dose GC also decreases tissue plasminogen activator activity (t-PA) and increases plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels increasing the procoagulant potential of GC. Inhibited angiogenesis, altered bone repair and nitric oxide metabolism can also result. Also, GC treatment modulates other vasoactive mediators such as endothelin-1, noradrenalin and bradykinin. Thus, GCs act as a regulator of local blood flow by modulating vascular responsiveness to vasoactive substances. Vasoconstriction induced in intraosseous femoral head arteries causes femoral head ischemia. GCs also cause ischemia through increased intraosseous pressure, which subsequently decreases the blood flow to the femoral head by apoptosis of ECs as well as elevating the level of adipogenesis and fat hypertrophy in the bone marrow. It is difficult to predict which patients receiving a specific dose of GC will develop ON, indicating individual differences in steroid sensitivity and the potential of additional mechanisms. The textbook model of ON is a multiple hit theory in which, with a greater number of risk factors, the risk of ON increases. While more effort is needed to better comprehend the role of GC in ON, newer data on GC action upon the endothelial cell and the regional endothelial bed dysfunction theory sheds new light on particular GC mechanisms. Better understanding of GC pathomechanisms can lead to better treatment options.

New insights into the pathogenesis of glucocorticoid-induced avascular necrosis: microarray analysis of gene expression in a rat model

IntroductionAvascular necrosis of the femoral head (ANFH) occurs variably after exposure to corticosteroids. Microvascular thrombosis is a common pathological finding. Since systemic thrombophilia is only weakly linked with ANFH, we propose that microvascular vessel pathology may be more related to local endothelial dysfunction and femoral head apoptosis. Corticosteroid effects on the endothelium and resultant apoptosis have been reported. We hypothesize that corticosteroids contribute to a differential gene expression in the femoral head in rats with early ANFH.MethodsBesides bone marrow necrosis, which is a common sign in ANFH and reported in the early stages, we include the presence of apoptosis in this study as a criterion for diagnosing early disease. Forty Wistar Kyoto (WKY) rats were randomized to either a corticosteroid-treated group or an age-matched control group for six months. After sacrifice, the femoral heads were examined for ANFH. Total mRNA was extracted from femoral heads. Affymetrix exon array (Santa Clara, CA, USA) was performed on 15 selected RNA samples. Validation methods included RT-PCR and immunohistochemistry (IHC).ResultsAlthough rat exon array demonstrated a significant upregulation of 51 genes (corticosteroid(+)/ANFH(+) VS control), alpha-2-macroglobulin (A2M) gene was particularly over-expressed. Results were validated by RT-PCR and IHC. Importantly, A2M is known to share vascular, osteogenic and cartilage functions relevant for ANFH.ConclusionsThe findings suggest that corticosteroid-induced ANFH in rats might be mediated by A2M. Investigation of A2M as a potential marker, and a treatment target, for early ANFH should be carried out.

Effect of high-dose dexamethasone on endothelial haemostatic gene expression and neutrophil adhesion

Glucocorticoid usage especially at high doses is complicated by adverse outcomes such as thrombotic events or acceleration of inflammatory response in conditions like myeloma and osteonecrosis. The mechanism(s) through which high-dose dexamethasone (HDDEXA) causes vascular injury remains unclear. We hypothesized that HDDEXA sensitizes endothelial cells (EC) to the effect of inflammatory mediators and modulates endothelial haemostatic gene expression and leukocyte adhesion. Human umbilical vein endothelial cells (HUVECs) were grown in the absence or presence of HDDEXA and were also tested in the presence or absence of tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) or thrombin. mRNA and protein expression were measured and the functional consequences of HDDEXA preconditioning on cell adhesion molecules (CAM) were determined by agonist-mediated leukocyte adhesion assay. Treatment with HDDEXA resulted in an increased induction of CAM, tissue factor and von Willebrand factor, while down-regulating thrombomodulin and urokinase. HDDEXA alone had no effect on adhesion but resulted in enhanced TNF-alpha- and LPS-mediated adhesion of neutrophils. Together, these findings suggest that HDDEXA sensitizes HUVEC to the effect of inflammatory mediators and induces a pro-adhesive environment in primary EC. This finding is of importance when glucocorticoid usage is required at therapeutic high doses in patients with or without thrombotic risk factors.

A rat model of early stage osteonecrosis induced by glucocorticoids

BackgroundGlucocorticoid (GC)-induced osteonecrosis (ON) is an important complication of medical therapy. The exact pathomechanisms of ON has not been clearly elucidated. There is a need for a reproducible animal model that better approximates the clinical scenario.MethodsTo determine the genetic susceptibility of rats to develop GC-induced femoral head ON, we evaluated 5 different inbred strains of rats (Spontaneous Hypertensive Rat, Wistar Kyoto, Wistar Furth, SASCO Fisher and Lewis). Prednisone pellets (dosage of 1.82-2.56 mg/kg/day) were implanted subcutaneously for 90. After 90 days, the femurs were resected and examined histologically and radiographically. Pathological and histological examination was performed. Hematoxylin and eosin (H & E) staining was used to delineate the femoral head osteonecrosis lesions as well as abnormalities of articular cartilage and growth plate.ResultsThe greatest differences in H & E staining were seen in the Wistar Kyoto and Wistar Furth groups. In these groups 4 out of 5 and 3 out of 5, respectively, steroid-induced rats revealed growth plate disruption with acellular areas. The TUNEL apoptosis staining assay for apoptosis revealed that 4 out of 5 of Wistar Kyoto rats, 5 out of 5 of Wistar Furth, 2 out of 4 of surviving Lewis and 2 out of 2 of the surviving spontaneous hypertensive rats had apoptotic osteocytes in trabeculae, whereas none of the Fisher rats showed apoptotic osteocytes.ConclusionsWe postulate that Wistar Kyoto, Wistar Furth and spontaneous hypertensive rats may be strains of rats more susceptible to develop ON of the femoral head while Fisher rats were the most resistant.

The PETALE study: Late adverse effects and biomarkers in childhood acute lymphoblastic leukemia survivors

Childhood cancer survivorship issues represent an established public health challenge. Most late adverse effects (LAEs) have been demonstrated to be time and treatment dependent. The PETALE study is a multidisciplinary research project aiming to comprehensively characterize LAEs and identify associated predictive biomarkers in childhood acute lymphoblastic leukemia (cALL) survivors.

Isolation and Characterization of Human Bone-Derived Endothelial Cells

Historically, the etiology of local bone pathologies, such as avascular necrosis, has been related to intravascular occlusion. Recent reports have highlighted the occlusion of arteries, venules, and/or capillaries in bone tissue. Endothelium of bone presumably participates locally in the formation of the microvascular thrombosis. It is also known that endothelial cells (ECs) play a central role in angiogenesis, a process seen in osteosarcoma, amongst other bone diseases. Given the well-recognized heterogeneity of ECs throughout the body, investigations of local bone disease related to endothelium processes may be more appropriately targeted on bone ECs rather than other primary ECs or an immortalized EC line. In the current study, mechanical and enzymatic methods are described to isolate ECs from cancellous human bone tissue followed by immunomagnetic bead separation to purify the cell populations. The human bone-derived endothelial cells (hBDECs) were characterized based on endothelial cell antigen expression and functional assays. This study is the first report of isolation and expansion of ECs from human bone tissue. Isolation of hBDECs in human vascular bone diseases may facilitate the study of the molecular and/or genetic abnormalities in the vasculature system that contributes to the initiation and/or progression of the disease.

Osteonecrosis of the femoral head: genetic basis

ABSTRACTPurposeGenetic factors and hereditary forms of osteonecrosis of the femoral head (ONFH) have been elucidated through genetic association studies. The significance of these cases is that they suggest an alternative hypothesis to the development of the disease. This review presents a summary of single nucleotide polymorphisms (SNPs) and other genetic mutation variations found in association with ONFH, including our recent identification of a novel mutation in the transient receptor potential vanilloid 4 (TRPV4) gene in association with inherited ONFH. The purpose of this review is to consolidate and categorize genetic linkages according to physiological pathways.MethodsA systematic review of literature from PubMed and Google Scholar was undertaken with a focus on genetic linkages and hereditary case studies of the disease. Recent genetic analysis studies published after 2007 were the focus of genetic linkages in non-hereditary cases.ResultsThe summary of these genetic findings identifies biological processes believed to be involved in the development of ONFH, which include circulation, steroid metabolism, immunity, and the regulation of bone formation.ConclusionTaken together, these associations may lead to new pathways of bone repair and remodeling while opening new avenues for therapeutic targets. Knowledge of genetic variations could help identify individuals considered to be at higher risk of developing ONFH and prevent the multiple hit effect.