Said Kamel

Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption

Several recent studies have suggested that flavonols, a class of phytochemicals with many biological activities, might exert a protective effect against post-menopausal bone loss. In the present study, we investigated the effects of quercetin and kaempferol, two of the major naturally occurring flavonols on the in vitro bone resorbing activity of osteoclasts. Our results indicate that both compounds, at concentrations ranging from 0.1 to 100 microM reduce bone resorption in a time and dose-dependent manner. Significant inhibitory effects were observed at concentrations as low as 0.1 microM especially with kaempferol. The IC(50)s, or concentration inhibitory of 50% of basal resorption, calculated for quercetin and kaempferol were 1.6 and 5.3 microM, respectively. Using highly purified rabbit osteoclasts, we showed that both flavonols directly induce apoptosis of mature osteoclasts in the same dose-range effective for inhibiting bone resorption. When osteoclasts were treated with 50 microM of quercetin and kaempferol, intracellular reactive oxygen species levels decreased significantly by 75 and 25%, respectively, indicating these molecules keep their antioxidant properties at this concentration. However, at concentrations below 50 microM, neither quercetin nor kaempferol exerted antiradical action, suggesting that antioxidant properties cannot fully explain the inhibitory effect on bone resorption. Finally, we report that kaempferol-, but not the quercetin-induced inhibition of bone resorption was partially abolished by the presence of the pure anti-estrogen ICI 182780 suggesting that kaempferols estrogenic effect could be involved in the inhibition of bone resorption. The present study demonstrates that flavonols widely distributed in human diet such as quercetin and kaempferol, exert a potent inhibitory effect on in vitro bone resorption.

Flavonoid quercetin decreases osteoclastic differentiation induced by RANKL via a mechanism involving NFκB and AP‐1

Flavonoids are micronutrients widely present in food of plant origin. They have been attributed pharmacological properties such as anticancer and prevention of age‐related pathologies. It has been recently hypothesized that flavonoids increase bone mass and prevent osteoporosis. However, little is known about the in vitro effects of flavonoids on osteoclast activities. We investigated the effects of quercetin, one of the most commonly occurring flavonoids, on osteoclast differentiation which is a critical determinant step of in vivo bone resorption. Two in vitro models of osteoclast differentiation were used in this study: a murine one, involving the culture of RAW 264.7 cells in presence of receptor activator of NFκB ligand (RANKL), and a human model consisting of differentiating peripheral blood monocytic cells (PBMC) isolated from peripheral blood in presence of RANKL and macrophage‐colony stimulating factor (M‐CSF). Osteoclastogenesis was assessed by osteoclast‐like number, tartrate resistant acid phosphatase (TRAP) activity, and bone resorbing activity. We showed that quercetin (0.1–10 μM) decreased osteoclastogenesis in a dose dependent manner in both models with significant effects observed at low concentrations, from 1 to 5 μM. The IC50 value was about 1 μM. Analysis of protein–DNA interaction by electrophoretic mobility shift assay (EMSA) performed on RAW cells showed that a pre‐treatment with quercetin inhibited RANKL‐induced nuclear factor kB (NFκB) and activator protein 1 (AP‐1) activation. NFκB and AP‐1 are transcription factors highly involved in osteoclastic differentiation and their inhibition could play an important role in the decrease of osteoclastogenesis observed in the presence of quercetin. In conclusion, the present results demonstrate for the first time that quercetin, a flavonoid characterized by antioxidant activities, is a potent inhibitor of in vitro osteoclastic differentiation, via a mechanism involving NFκB and AP‐1.

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.

Calcimimetics increase CaSR expression and reduce mineralization in vascular smooth muscle cells: mechanisms of action

AIMS Vascular calcification (VC) contributes to morbidity and mortality in patients with chronic kidney disease (CKD). Allosteric modulators of the calcium (Ca)-sensing receptor (CaSR) may slow the progression of VC in CKD patients either by reducing serum parathyroid hormone (PTH), Ca, and phosphate levels or by a direct effect on the vessel wall. The aim of this study was to examine the effects of calcimimetics on CaSR expression, cell phenotype, and mineral deposition in human vascular smooth muscle cells (h-VSMCs). METHODS AND RESULTS Primary h-VSMCs were exposed for 14 days to increasing concentrations of Ca(2+) (from 1.8 to 5 mmol/L) in the presence or absence of calcimimetics R-568 or AMG 641 (0.1 μmol/L). Mineralization was detected by Alizarin red staining, and the cell phenotype was assessed using immunocytochemistry and qRT-PCR. CaSR expression was evaluated using flow cytometry. Short- and long-term exposure (1 day to 14 days) of h-VSMCs to calcimimetics promoted CaSR protein transport from the endoplasmic reticulum to the plasma membrane with enhanced CaSR expression on the cell surface, together with an increase in total cell CaSR expression due to enhanced biosynthesis. In pro-mineralizing conditions, exposure to calcimimetics counteracted the Ca(2+)-dependent reduction of CaSR expression, decreased matrix collagen secretion, and mineral deposition by ~90%. These effects involved CaSR activation since it could be inhibited by CaSR siRNA, but not scrambled siRNA. CONCLUSIONS The calcimimetic-dependent increase in biosynthesis and activation of the CaSR in h-VSMCs probably play a key role in the protection against calcium-induced VC.

Involvement of capacitive calcium entry and calcium store refilling in osteoclastic survival and bone resorption process.

Bone resorption is closely dependent on osteoclastic survival and osteoclast apoptotic cell death could represent a key step at the end of this process. In order to precise the possible role of calcium movement in osteoclastic cell death, we investigated whether intracellular calcium store replenishment and capacitive calcium entry (CCE) are involved in osteoclastic survival and bone resorption. We demonstrate that (i). thapsigargin, a sarco-endoplasmic reticulum calcium ATPase pump (SERCA) blocker, decreases both osteoclastic survival and bone resorption process, (ii). 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365, two store-operated channel (SOC) blockers, dramatically decrease osteoclastic survival and bone resorption and (iii). culture in calcium-free medium and thapsigargin exposure synergically inhibit osteoclastic survival which falls dramatically to a value close to 0% (P<0.001). Inversely, osteoclastic survival increases significantly when thapsigargin-treated cells are cultured in the presence of 20mM calcium, suggesting that increasing extracellular calcium concentration stimulates osteoclasts survival when the filling of intracellular stores is prevented. Taken together, our data strongly suggest that in osteoclasts, calcium movements between cellular compartments involved in the regulation of calcium signalling, such as calcium stores refilling and CCE, are closely associated to the regulation of osteoclast survival and bone resorption.

Tumor necrosis factor-related apoptosis-inducing ligand and vascular calcification.

Vascular calcification is frequent in patients with chronic kidney disease. Osteoprotegerin (OPG, a soluble factor which blocks osteoclast differentiation) has recently been implicated in the genesis of vascular calcification. Given that OPG can bind the pro‐apoptotic tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), we hypothesized that the TRAIL protein is involved in the formation of vascular calcification both in vitro and in vivo. Using an immunohistochemical approach, we evaluated TRAIL and OPG expression on aortic valves slides from non‐uremic and uremic wild type and apolipoprotein knockout (Apo E−/−) mice. We also tested the in vitro effects of TRAIL on cultured primary human vascular smooth muscle cells (hVSMC). We further assayed serum soluble TRAIL (sTRAIL) levels in hemodialysis patients and correlated them with vascular calcification scores. Our results demonstrated that: (i) TRAIL and OPG were expressed inside the atheroma plaque in non‐uremic ApoE−/− mice, but not in wild type mice; and (ii) uremia enhanced the expression levels. TRAIL enhanced the phosphate‐induced mineralization of hVSMCs in a dose‐dependent manner. In clinical terms, we demonstrated that sTRAIL is depressed in the sera of hemodialysis patients, but was not correlated with vascular calcification. Our results suggest that TRAIL may be involved in the formation of vascular calcification in certain experimental settings; however, its role in chronic kidney disease patients requires further evaluation.

Oligogalacturonic acid Inhibit Bone Resorption and Collagen Degradation through its interaction with type I Collagen

In this study, we showed that oligogalacturonic acid (OGA) purified from flax pectin inhibit in vitro osteoclastic bone resorption in a dose-dependent manner. The OGA inhibitory effect was neither linked to an effect on osteoclast apoptosis, nor to an inhibition of cathepsin K activity. By means of an in vitro collagen degradation assay we demonstrated that OGA prevented triple-helical type I collagen cleavage by cathepsin K in a dose and chain length dependent manner. This inhibition was not restricted to cathepsin K, since collagenolytic activity of other lysosomal cysteine proteases, such as cathepsin B and cathepsin L, as well as matrixmetalloproteinases such as MMP-9 were also inhibited. Interestingly, using non-collagen substrates we demonstrated that OGA does not inhibit the proteolytic activity of cathepsin B and L, suggesting that OGA inhibits collagen degradation without affecting the lysosomal cysteine enzyme proteolytic activity. Finally, preliminary study using surface plasmon resonance (SPR) showed that OGA binds to type I collagen but not to albumin, consistent with a specific effect on collagen. These results suggest that the observed inhibition of collagen degradation by OGA may be due to its ability to bind to the collagen molecule. By masking the collagen surface, OGA may render the collagen cleavage site less accessible to enzymes and thus prevent its enzymatic degradation.

Determination and Modulation of Total and Surface Calcium-Sensing Receptor Expression in Monocytes In Vivo and In Vitro

Expression of the calcium-sensing receptor (CaSR) has previously been demonstrated in human circulating monocytes (HCM). The present study was designed to measure CaSR expression in HCM and to examine its potential modulation by pro-inflammatory cytokines, Ca2+, vitamin D sterols in U937 cell line. Twenty healthy volunteers underwent blood sampling with subsequent isolation of peripheral blood mononuclear cells (PBMC) at 3 visits. Flow cytometry analysis (FACS) was performed initially (V1) and 19 days later (V2) to examine intra- and intersubject fluctuations of total and surface CaSR expression in HCM and 15 weeks later (V3) to study the effect of vitamin D supplementation. In vitro experiments were conducted to assess the effects of pro-inflammatory cytokines, calcidiol, calcitriol and Ca2+ on CaSR expression in U937 cell line. By FACS analysis, more than 95% of HCM exhibited cell surface CaSR staining. In contrast, CaSR staining failed to detect surface CaSR expression in other PBMC. After cell permeabilization, total CaSR expression was observed in more than 95% of all types of PBMC. Both total and surface CaSR expression in HCM showed a high degree of intra-assay reproducibility (<3%) and a moderate intersubject fluctuation. In response to vitamin D supplementation, there was no significant change for both total and surface CaSR expression. In the in vitro study, U937 cells showed strong total and surface CaSR expression, and both were moderately increased in response to calcitriol exposure. Neither total nor surface CaSR expression was modified by increasing Ca2+ concentrations. Total CaSR expression was concentration dependently decreased by TNFα exposure. In conclusion, CaSR expression can be easily measured by flow cytometry in human circulating monocytes. In the in vitro study, total and surface CaSR expression in the U937 cell line were increased by calcitriol but total CaSR expression was decreased by TNFα stimulation.

Troubles minéraux et osseux de la maladie rénale chronique (TMO-MRC)

Resume Le maintien de la calcemie et de la phosphatemie dans des valeurs etroites est le resultat d’une regulation complexe faisant intervenir des echanges entre le rein, l’intestin et le squelette. Le rein joue un role essentiel dans cette regulation en permettant a court terme un maintien de l’equilibre phosphocalcique grâce a une adaptation de l’elimination renale du calcium et des phosphates aux apports nutritionnels. Cette adaptation est sous la dependance d’hormones dont la forme active de la vitamine D et l’hormone parathyroidienne. Notre comprehension de la regulation du metabolisme phosphocalcique s’est considerablement enrichie et complexifiee ces dernieres annees grâce a la decouverte de nouveaux acteurs dont le FGF23 et Klotho. La diminution progressive de la fonction renale telle qu’elle est observee dans la maladie renale chronique (MRC) s’accompagne de troubles mineraux et metaboliques importants regroupes sous le terme de « troubles mineraux et osseux de la maladie renale chronique ou CKD-MBD pour « chronic kidney disease - mineral and bone disorders ». Ces troubles sont caracterises par des anomalies biochimiques du metabolisme phosphocalcique, du remodelage et de la mineralisation osseuse et par le developpement de calcifications vasculaires. L’hyperparathyroidie secondaire a l’insuffisance renale chronique, dont les mecanismes sont aujourd’hui en passe d’etre mieux compris et dans lequel la retention de phosphate joue un role important, est un determinant physiopathologique essentiel dans la survenue des differentes anomalies des tissus osseux et des tissus mous. Des travaux recents ont aussi demontre que les deregulations du metabolisme des phosphates et du calcium sont fortement associees a la mortalite et la morbidite cardiovasculaire du patient ayant une insuffisance renale chronique. Dans cette revue, apres un rappel sur la regulation de la calcemie et de la phosphatemie, nous decrirons dans un premier temps les mecanismes physiopathologiques a l’origine des troubles mineraux de la MRC et dans un second temps les consequences de ces troubles mineraux sur le squelette et la paroi vasculaire. La biologie tenant une place importante dans le diagnostic et le suivi therapeutique de ces atteintes, nous rappellerons les principales recommandations emises par la fondation internationale « Kidney disease improving global outcome (KDIGO) » sur les parametres biologiques a mesurer.

AB0128 Total Calcium-Sensing Receptor Expression in Circulating Monocytes is Increased in Rheumatoid Arthritis Patients with Severe Coronary Artery Calcification

Background Human circulating monocytes express the calcium-sensing receptor (CaSR) and are involved in atherosclerosis. Vascular calcification is commonly used as a subclinical marker of atherosclerosis and has been linked to increased all-cause mortality, cardiovascular mortality and coronary events. Moreover, patients with RA are known to develop early-onset, widespread calcification in various vascular beds. We have previously assessed CaSR expression by flow cytometry in human circulating monocytes and have provided evidence that this type of approach could be useful in certain clinical situations, in which changes in CaSR expression can be expected. Objectives This study investigated the potential association between vascular calcification in rheumatoid arthritis (RA) and CaSR expression in circulating monocytes. Methods In this cross-sectional study, 50 RA patients were compared to 25 control subjects matched for age and gender. Isolation of peripheral blood mononuclear cells and flow cytometry analysis were performed to study the surface and total CaSR expression in circulating monocytes. Coronary artery calcium (CAC) and abdominal aortic calcification (AAC) scores were evaluated by computed tomography and an association between these scores and the surface and/or total CaSR expression in circulating monocytes in RA patients was investigated. Results The two groups were similar in terms of age (RA: 60.9±8.3 years, versus controls: 59.6±5.3 years) and gender (RA: 74.0% females versus 72.0% females). RA patients did not present a higher prevalence or a greater burden of CAC or AAC compared to age- and gender-matched controls. When compared with control subjects, RA patients did not exhibit greater total CaSR (101.6% ±28.8 vs. 99.9% ±22.0) or surface CaSR (104.6% ±20.4 vs. 99.9% ±13.7) expression, but total CaSR expression in circulating monocytes was significantly higher in RA patients with severe CAC (Agatston score ≥200, n=11) than in patients with mild-to-moderate CAC (1-199, n=21) (p=0.01). Conclusions This study demonstrates, for the first time, that total CaSR expression in human circulating monocytes is increased in RA patients with severe coronary artery calcification. References Paccou J, Brazier M, Mentaverri R, et al. Vascular calcification in rheumatoid arthritis: prevalence, pathophysiological aspects and potential targets. Atherosclerosis 2012;224:1418-23. Paccou J, Boudot C, Mary A, et al. Determination and modulation of total and surface calcium-sensing receptor expression in monocytes in vivo and in vitro. PLoS one 2013;8:e74800. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2377