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Bone loss in critical illness: from molecular pathways to in vivo analysis.

Bone loss in critical illness: from molecular pathways to invivo</> analysis</>

Advances in intensive care medicine have enabled patients to survive life-threatening insults through mechanical and/or pharmacological vital organ support. However, approximately 30% of all patients do not recover from these acute conditions within a few daysand evolve towards a phase of prolonged critical illness. This chronic phase is a severe therapy-resistant catabolic state that is hallmarked by lean tissue wasting of skeletal muscle, reduced neuro-endocrine stimulation of target endocrine organs and non-resolving multiple organ failure. Critically ill patients also display marked alterations in calcium and bone metabolism. From admission, many patients display hypocalcemia and circulating levels of bone formation markers are decreased, suggestingimpaired osteoblast maturation. Bone resorption markers are increased 6- to 15-fold in critically ill patients and aggravate during stay, suggesting increased osteoclast activity. Since classic metabolic bone diseases such as postmenopausal osteoporosis are characterized by a 2-fold increase in bone resorption, these findings are remarkable. This imbalance hints at an uncoupling between bone breakdown and bone formation that may result in bone loss during critical illness and could predispose critically ill patients to inefficient traumatic or surgical fracture healingand osteoporosis. Recently, a retrospective longitudinal case-cohort study revealed an increased risk of fragility fractures in elderly female survivors of the intensive care unit, suggesting an association between critical illness and skeletal morbidity. 

The general aim ofthis PhD project was to gain insight into the effect of critical illness on bone metabolism and search for possible underlying mechanisms. We hypothesized that critical illness causes an impairment of osteoblast differentiation and an increase in osteoclast formation and activity, leading to an uncoupling between bone formation and degradation and subsequently causing bone loss. 

In the first study, we analyzed the effect of critical illness on bone physiology at the cellular level. Critically ill patients revealed an increased number of circulating early osteoclast precursors displaying increased osteoclastogenic potential, which resulted in increased osteoclast formation in vitro</>, bothin the presence and absence of osteoclastogenic factors. Humoral factors present in the serum from critically ill patients further potentiated osteoclast formation and activity from patient peripheral blood mononuclear cells. In critically ill patients, serum concentrations of inflammatory cytokines were significantly higher than in healthy controls. In contrast to classic metabolic bone diseases such as rheumatoid arthritis, we found that neutralizing these cytokines in critically ill patient serum in vitro</> did not inhibit osteoclast formation in patient cells, suggesting that despite the major increase in inflammatory cytokines, other factors in the serum are involved. Blocking signaling through an immunoreceptor inhibited osteoclast formation in the presence and absence of cytokine neutralizing antibodies. These data suggest the involvement of humoral factors and/or immunomodulatory mechanisms acting throughimmunoreceptors from an alternative signaling pathway in aberrant osteoclastogenesis during critical illness, independent of cytokine signaling. 
To study the effect of critical illness on osteoblast formation and activity in vitro</>, healthy human periosteal derived cells were grown in either healthy or patient serum. We found an increase in proliferation and migration activity of the cells treated with patient serum. Additionally, an 'in vivo</>' murine model for bone formation revealed significantly less mature bone formation in calcium phosphate scaffolds seeded with patient serum-treated cells, suggesting impaired osteoblast activity during critical illness. Furthermore, we found a decrease in angiogenesis markers in the patient serum-treated cells in vitro</>, which was confirmed in the in vivo</> murine model of bone formation, where a reduction in vascularization was observed. These studies suggest that at the cellular level, critical illness may induce increased osteoclast formation and activity, possibly mediatedthrough alternative immunoreceptor signaling in addition to decreased bone formation and angiogenesis. 

In the second part of this project, we attempted to confirm these results at the tissue level in our validated rabbit model of prolonged (7 days) critical illness. Critically ill rabbits display hypocalcemia and a decrease in a mature osteoblast marker in plasma, both of which are also observed in human criticallyill patients. Sick rabbits also showed a significant reduction in trabecular bone area and bone mineral content and density. However, surprisingly, the decrease in trabecular bone could not be associated with an increase in the number of osteoclasts in trabecular bone sections or a change in the canonical marker of osteoclast activity in the sick rabbits. However, there was a significant increase in immunoreceptors from an alternative osteoclast signaling pathway. We also found a significant reduction in markers of osteoblast differentiation and angiogenesis in the trabecular bone of critically ill rabbits. This in vivo</> study confirmed the results of our previous in vitro</> studies at the tissue level, and suggests that the observed critical illness-related reduction in trabecular bone may result from osteoclastic bone hyperresorption mediated through an alternative signaling pathway rather than the canonical RANKL pathway, combined with a decrease in osteoblast formation and angiogenesis.

The mechanisms responsible for changes in bone metabolism during prolonged critical illness are still largely unknown, but are most likely multifactorial. In the third and fourth study, we investigated two potential underlying mechanisms of aberrant osteoclastogenesis during critical illness. 
The importance of autophagy for normal osteoclastogenesis has become apparent in Paget's disease where amutation in an autophagy cargo receptor results in aberrant osteoclastogenesis and focal bone loss. In critically ill patients, deficient autophagy was found in liver, kidney and skeletal muscle. Therefore, we studied the role of autophagy in critical illness-induced aberrant osteoclastogenesis in vitro</>. Osteoclast differentiation markers were increased in patient peripheral blood mononuclear cells compared to healthy cells, while autophagy markers were decreased. Additionally, the accumulation of autophagic cargo receptor in patient cells in the absence of a rise in gene expression supports the hypothesis of deficient autophagyin osteoclasts during critical illness. Pharmacological induction of autophagy reduced osteoclast formation in patient cells in vitro</> and in vivo</> by protecting critically ill rabbits against trabecular and cortical bone loss after only 3 days of illness. In addition, osteogenic markers were increased towards healthy levels and osteoclastogenic immunoreceptors were reduced. In vitro</>, we showed global hypomethylation in patient cells and osteoclasts, which could possibly be linked with deficient autophagic signaling during critical illness-induced aberrant osteoclastogenesis, as chromatin remodeling is known to beinvolved in the epigenetic regulation of key regulators of autophagy. 

Emerging evidence of the association between dysregulation of microRNAs, which is another form of epigenetic regulation, and increased osteoclast activity in bone diseases such as rheumatoid arthritis, suggests that microRNAs may also play a role in osteoclastogenesis during critical illness. Genome-wide gene expression analysis has revealed thatthere are many differences in gene expression patterns between healthy and critically ill patient osteoclasts. Immune-related signaling seems to play a key role in osteoclast formation, along with epigenetic signaling, and a number of downregulated genes supported our hypothesis of deficient autophagy in critical illness.

In conclusion, our data revealed an increase in osteoclast formation and activity, together with a reduction in bone formation and angiogenesis at the cellular and tissue level. The aberrant osteoclastogenesis observed is not likely to be stimulated through the classical RANKL osteoclastogenic pathway, but via immunoreceptors from an alternative pathway, independent of cytokine signaling. A possible underlying mechanism for critical illness-induced aberrant osteoclastogenesis is deficient autophagy, which may be caused by global histone hypomethylation. Microarray analysis of gene and microRNA expression profiles supported the hypotheses concerning the involvement of immune-related and epigenetic signaling, as well as deficient autophagy in aberrant osteoclastogenesis during critical illness. 
These data provide insights into the underlying mechanisms of this therapy-resistant catabolism and open perspectives for therapies that activate autophagy or target specificgenes or microRNAs in order to reduce increased osteoclast formation and activity in critically ill patients. 

Date:1 Nov 2009 →  9 Dec 2013
Keywords:Critically ill children, Metabolism, Bone growth
Disciplines:Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences
Project type:PhD project