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Study of the role of apolipoprotein E (apoE), a key component of the lipid and lipoprotein transport system, in the development of obesity-related osteoarthritis

Final Report Summary - APOEOSTEOARTHRITIS (Study of the role of apolipoprotein E (apoE), a key component of the lipid and lipoprotein transport system, in the development of obesity-related osteoarthritis)

FINAL REPORT
Osteoarthritis (OA) is a very common degenerative joint disease that accounts for a considerable proportion of disability in adults. The aetiology of OA is multifactorial, inasmuch as increased mechanical stimulation, trauma, metabolic factors and genetic susceptibility contribute to its pathogenesis. Definitely, one of the most common conditions that lead to OA is obesity. Excess body weight is associated with elevated mechanical stimulation of the weigh-bearing joints that leads to cartilage destruction and ultimately OA development. However, more resent data have demonstrated that non-mechanical, metabolic factors have also central role in the development of OA in obese people. For instance, relatively recent molecular and biochemical studies have highlighted the pivotal role of adipokines, such as leptin, adipnectin, resistin in OA pathobiology. This hypothesis is further supported by a considerable volume of reports having shown that OA is tightly associated with cardiovascular-related conditions, such as hypertension, hypercholesterolemia, abdominal obesity, dyslipidemia, type 2 diabetes and metabolic syndrome, which are coupled to distorted lipid metabolism. Clearly, the need to understand the biological mechanisms that govern the pathogenesis and progression of OA and to develop novel therapeutic strategies to effectively treat this common disease is becoming increasingly pressing.
Triggered by these data the central hypothesis of this proposal is that apolipoprotein E (apoE), which is a key component of the chylomicron/VLDL and HDL pathways and promotes diet-induced obesity, may also contribute to the pathogenesis and progression of obesity-related osteoarthritis (OA).
Significant results of the project:
i) The deficiency of the cardinal components of the lipid and lipoprotein transport system, namely LCAT and ApoA-I leads in the development of OA in mice after long exposure to high-fat (Western-type) diet.
Our morphological, molecular and biochemical analyses revealed that ApoE is not associated with the development of OA with or without long-tem exposure to Western-type diet (WTD). Recent epidemiological studies have demonstrated that HDL is decreased in the serum of OA patients compared to non-OA individuals, clearly suggesting that HDL plays a central role in the pathogenesis of OA. Moreover, it has been recently documented that lipid droplets accumulate within osteoarthritic chondrocyte due to impaired expression and function of cholesterol efflux molecules (namely ApoA-I and Liver X Receptors) resulting in damage of articular cartilage in OA. Driven by these results we then decided to ask whether the other vital components of the lipid and lipoprotein transport system, namely Lecithin—cholesterol acyltransferase (LCAT) and apolipoprotein A-I (ApoA-I) that are involved in HDL maturation have a role in the pathobiology of OA. For this reason we used LCAT and ApoA-I deficient mouse models. Interestingly, we showed for the first time that both the LCAT and the ApoA-I deficient mice developed ΟΑ after the consumption of WTD. This finding suggests that LCAT and ApoA-I deficiency and hence the inability to produce mature HDL, predisposes to the development of OA in mice following chronic insult by WTD. More importantly, even though LCAT-/- mice fed WTD were obese and significantly heavier than their wild-type counterparts, the ApoA-I-/- mice were not obese a finding that implies that OA development in apoA-I-/- mice is due to the lack of functional HDL, rather than to mechanical stimulation imposed by their body weight. This is a very significant result since it suggests for the first time that formation of mature HDL protects against diet-induced OA. Furthermore, the causative role of HDL in the pathogenesis of OA that we have identified with the present experimental set-up may also explain the increased risk of patients with OA towards cardiovascular disease.
ii) The LCAT and the apoA-I deficient mice fed WTD had significantly elevated number of adipocytes in their bone marrow as compared to the control groups.
We hypothesize that since bone marrow fat functions in a fashion similar to white adipose tissue it may produce and secrete adipokines (e.g. leptin, adiponectin, resistin) that may have an effect on articular cartilage and thus lead to the development of OA. This is a novel and potentially significant hypothesis that further adds to our effort to illuminate the role of lipid/fat metabolism in the pathogenesis of osteoarthrtis. Our research team has planned further in vitro studies to explore this hypothesis.
iii) ApoE deficiency predisposes to reduced bone mass and to osteoporosis in mice following long-term exposure to WTD, affecting both osteoblast and osteoclast function.
This finding was based on both static (micro-CT) and dynamic (calcein labeling and TRAP staining) histomorphometry that were performed in the 1st period of the project. Interestingly, in ApoE-/- mice the osteoclastic activity was elevated, while the osteoblastic function was decreased as determined by TRAP staining and calcein labeling, respectively. This is another interesting finding of the present project since it suggests that apoE protects towards the development of osteoporosis (OP) after the consumption of the common WTD.
OP is a very common metabolic condition characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. It is nowadays well established that OP constitutes a major, gradually increasing public health, social and financial problem worldwide. The past few years many researchers have focused on the role of molecules implicated in fat metabolism in the regulation of osteoblast and osteoclast function and consequently in the pathogenesis of OP. In the same line, in our effort to further investigate this novel finding and to uncover the corresponding molecular mechanism that links apoE with OP, we performed a series of “sophisticated” molecular and biochemical experiments on mice bone marrow mesenchymal stem cells and whole bone marrow cells obtained from experimental mice femora. We examined several genes related to osteoblastogenetic and osteoclastogenetic functions and found that osteoblatst activity was significantly reduced, while osteoclast activity was remarkably elevated in the apoE KO compared to WT mice. Notably, since the calcification markers/regulators osteocalcin and osteonectin did not display differences between the groups we propose that apoE deficiency affects the early stages of osteoblast biology. Not surprisingly, since the histological sections of apoE deficient mice femora were devoid of adipocytes, the expression of the lipoblastic regulators C/EBP and PPARγ was reduced compared to WT. This implies that apoE deficiency displays specificity for both the osteoblastic and the lipoblastic primary events after exposure to WTD. In symphony with our in vitro and histomorphometry data, RAMAN spectroscopy revealed that the collagen cross-links ratio was significantly reduced in the apoE KO mice femora compared to the WT, predisposing to fractures.
iv) Deficiency of the major regulator of HDL biogenesis Apolipoprotein A-I, is strongly associated with the development of osteoporosis in mice.
Having shown that ApoE is implicated in the pathogenesis of OP, while HDL impairment results in OA in mice fed WTD, in the next set of experiments we aimed at answering the question whether the reduction and/or the absence of HDL is also involved in OP pathobiology. For this reason we performed static (microCT) and dynamic (TRAP stain, calcein labeling) histomorphometry of lumbar vertebrae from apoA-I-/- and WT (C57BL/6) mice and found that apoA-I deficiency results in severe bone mass reduction. Interestingly, further analysis with RAMAN spectroscopy and three-point-bending studies on mice femora showed that bone mass reduction in the apoA-I-/- was accompanied by significant reduction in collagen cross-links as well as mechanical stress, elasticity and max load to fracture.
v) ApoA-I deficiency results in impaired osteoblastic but not in increased osteoclastic function.
Next, we explored the mechanisms that regulate bone mass in the apoA-I KO mice. Using an elegant set of in vitro molecular experiments on MSC, osteoblats and osteoclasts derived from mice femur bone marrow we found the apoA-I deficiency primarily results in significant reduction of genes that regulate the function of osteoblasts. Importantly, the number and function of the bone resorbing cells, osteoclats remains unaffected. Moreover, since the fat-related genes that we examined did not display differences in their expression pattern and levels between the groups we jumped to the conclusion that (ApoA-I deficiency exhibits high specificity for the osteoblast primary differentiation event. Finally, since LCAT deficiency does not affect bone mass we are allowed to hypothesize that it is HDL biosynthesis and not HDL maturation that affects bone cells, resulting in OP.

Our proposed models and hypotheses regarding the role of HDL perturbations in the pathogenesis of OA and OP are schematically presented in Figures 1 and 2, respectively (see attached file).
Collectively, our novel findings propose a functional cross-talk between HDL metabolic pathway and bone biology, further supporting the notion that bone diseased have a strongly have a lipid-related facet in their aetiopathogenesis. Moreover, these results raise the intriguing possibility that expression of therapeutic forms of apoA-I by gene therapy approaches may have a beneficial effect on OP treatment.
iv) Deficiency of ApoE, apoA-I and LCAT is implicated in diet-induced non-alcoholic fatty liver disease in mice.
In order to expand the current knowledge on the functions of HDL, we also examined ΚΟ mice to assess the involvement of apoE, apoA-I and LCAT in diet-induced accumulation of triglycerides in hepatocytes and its potential role in the treatment of nonalcoholic fatty liver disease (NAFLD). Notably, we found that that in addition to their properties in skeletal diseases, these molecules are important modulators of processes associated with diet-induced hepatic lipid deposition and NAFLD development in mice. Our findings are noveand may open the way for the development of new therapeutic interventions against fatty liver disease and probably liver cirrhosis (see template A - List of Publications: Karavia et al, FEBS J, 2011; Karavia et al, Mol Med 2012; Karavia et al, J Nutr Biochem 2013).

Potential impact and use:
The significance of the present research proposal is that it is the first to define the role of key-components of the chylomicron and HDL pathway, in the development of the 2 most common diseases OA and OP. Moreover, this study is the first to investigate thoroughly the functional link between the lipid and lipoprotein transport system and obesity-related degenerative and metabolic bone diseases using a unique collection of animal models. It is expected that the proposed project will provide important mechanistic insights in the development of the aforementioned pathologies. Furthermore, we expect that the proposed studies will reveal novel therapeutic agents and targets for their treatment. Such agents may be coupled with conventional technologies or the emerging nanotechnology or gene therapy methodologies, for the prevention and efficient treatment of these very common and potentially serious skeletal diseases. Finally, our findings pave the way towards the identification of novel molecular mechanisms that link HDL metabolic pathways and bone-related devastating neoplastic conditions such as hematopoietic malignancies and bone metastases.
The work and academic progress and achievements of the fellow have been acknowledged by the international scientific society, since he has been invited to give talks/lectures in well-respected congresss and seminars and he has also been invited as editor and guest editor in high-impact publications.
The results of the present proposal, the related publications and dissemination activities (Workshops, public speech) are available through the website: www.osteonet.gr