Extracellular pH: a fundamental regulator of bone cell function.

Systemic acidosis is associated with bone loss and impaired bone mineralisation. The aim of this PhD project was to further investigate the action of extracellular pH on the function of osteoclasts and osteoblasts. I showed that blood-derived human osteoclasts exhibited a highly reproducible acid-activation response, with maximal activation close to pH 7.0, and little activity at blood pH (7.4). These experiments also provided strong evidence that accessory cells, such as osteoblasts or stromal cells, are not required for acid-activation of resorption. The pH-activation profile of human osteoclasts was similar to that of the recently discovered KT-sensing human G- protein-coupled receptor OGR1. Expression of OGR1 and TDAG8 (another GPCR) was detected in human osteoclasts and was upregulated by low pH. I obtained evidence that the multifunctional receptor TRPV1, which senses protons, heat and capsaicin, was expressed by human osteoclasts and was also upregulated at pH 7.0. Moreover, I showed that the alkaloid capsaicin strongly stimulated osteoclasts in non- acidified conditions. To date, only pertussis toxin has been reported to activate osteoclasts without co-stimulation by H. Using mouse bone organ cultures I found that resorption-associated factors TRACP, cathepsin K and TRAF-6 were also upregulated by acidosis. The effect of PTH on human osteoclasts was also studied. I showed that PTH directly stimulates human osteoclasts in the absence of osteoblasts, but only when acid-activated. This finding suggests that the dogma that PTH stimulation of osteoclast is osteoblast-mediated may not be correct. Studies using primary rat osteoblast cultures showed that the formation of mineralised bone nodules is inhibited by acidosis. The same pH reduction, which increases Ca2+ and PO43" solubility of hydroxyapatite by 2- and 4-fold respectively, did not alter collagen production or osteoblast proliferation but decreased alkaline phosphatase activity and expression. Thus, the primary effect of acidosis on osteoblast function is to cause a selective inhibition of bone mineralisation. In conclusion, this study showed that the important "double negative" action of acidosis on bone cells is consistent with a pathophysiological role of bone as a reserve of base to buffer excess protons when the kidneys and lungs are unable to maintain acid-base balance within narrow physiological limits

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation

The effect of bone marrow microenvironment on the functional properties of the therapeutic bone marrow-derived cells in patients with acute myocardial infarction

<p>Abstract</p> <p>Background</p> <p>Treatment of acute myocardial infarction with stem cell transplantation has achieved beneficial effects in many clinical trials. The bone marrow microenvironment of ST-elevation myocardial infarction (STEMI) patients has never been studied even though myocardial infarction is known to cause an imbalance in the acid-base status of these patients. The aim of this study was to assess if the blood gas levels in the bone marrow of STEMI patients affect the characteristics of the bone marrow cells (BMCs) and, furthermore, do they influence the change in cardiac function after autologous BMC transplantation. The arterial, venous and bone marrow blood gas concentrations were also compared.</p> <p>Methods</p> <p>Blood gas analysis of the bone marrow aspirate and peripheral blood was performed for 27 STEMI patients receiving autologous stem cell therapy after percutaneous coronary intervention. Cells from the bone marrow aspirate were further cultured and the bone marrow mesenchymal stem cell (MSC) proliferation rate was determined by MTT assay and the MSC osteogenic differentiation capacity by alkaline phosphatase (ALP) activity assay. All the patients underwent a 2D-echocardiography at baseline and 4 months after STEMI.</p> <p>Results</p> <p>As expected, the levels of pO₂, pCO₂, base excess and HCO₃were similar in venous blood and bone marrow. Surprisingly, bone marrow showed significantly lower pH and Na⁺and elevated K⁺levels compared to arterial and venous blood. There was a positive correlation between the bone marrow pCO₂and HCO₃levels and MSC osteogenic differentiation capacity. In contrast, bone marrow pCO₂and HCO₃levels displayed a negative correlation with the proliferation rate of MSCs. Patients with the HCO₃level below the median value exhibited a more marked change in LVEF after BMC treatment than patients with HCO₃level above the median (11.13 ± 8.07% vs. 2.67 ± 11.89%, P = 0.014).</p> <p>Conclusions</p> <p>Low bone marrow pCO₂and HCO₃levels may represent the optimal environment for BMCs in terms of their efficacy in autologous stem cell therapy in STEMI patients.</p

Serum amyloid A inhibits RANKL-induced osteoclast formation

When mouse bone marrow-derived macrophages were stimulated with serum amyloid A (SAA), which is a major acute-phase protein, there was strong inhibition of osteoclast formation induced by the receptor activator of nuclear factor kappaB ligand. SAA not only markedly blocked the expression of several osteoclast-associated genes (TNF receptor-associated factor 6 and osteoclast-associated receptor) but also strongly induced the expression of negative regulators (MafB and interferon regulatory factor 8). Moreover, SAA decreased c-fms expression on the cell surface via shedding of the c-fms extracellular domain. SAA also restrained the fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by the well-known SAA receptors (formyl peptide receptor 2, Toll-like receptor 2 (TLR2) or TLR4). These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis and suggest that SAA is an important endogenous modulator that regulates bone homeostasis.open

A neglected requirement for optimizing treatment of age-related osteoporosis: Replenishing the skeleton’s base reservoir with net base-producing diets

Osteoporosis is a disorder of bone in which the mass of the bone is reduced and the bone's architecture at the microscopic level is disordered. Together those abnormalities predispose affected individuals to experience fractures despite only minimal trauma (i.e., fragility fractures). Age related osteoporosis is a common type of osteoporosis that occurs with aging in both men and women usually beginning after the age of peak bone mass. Research has found that the disorder can be partially reversed by reducing the net amount of acid that is produced when consuming typical Western diets. However, the amelioration that results has not been so dramatic or so consistent that physicians have adopted the procedure as part of the standard treatment for age-related osteoporosis. We propose that reducing the net acid load from the diet is not sufficient to reverse age related osteoporosis because it fails to supply base needed to restore the large amount of base in bone that had been lost by reacting with the net acid load of the diet that had been consumed for years or decades. Reducing the net acid load from the diet might be expected to have little ameliorative effect or merely slow the progression of the disorder. We hypothesize that both to restore osteoporotic bone to, or nearly to, its pre-disease state, as well as to eliminate the risk of fragility fractures, requires consuming diets that produce net amounts of base to restore the base lost from years to decades of consuming diets that produce net amounts of acid. We hypothesize also that the excess base and attendant subclinical metabolic alkalosis will both stimulate the cellular process of bone formation and suppress the cellular process of bone resorption, and thereby implement the restorative process

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis

The proton-activated ovarian cancer G protein-coupled receptor 1 (OGR1) is responsible for renal calcium loss during acidosis.

Hypercalciuria is a common feature during metabolic acidosis and associates to nephrolithiasis and nephrocalcinosis. The mechanisms sensing acidosis and inducing increased urinary calcium excretion are still unknown. Here we tested whether mice deficient for proton-activated Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) have reduced urinary excretion of calcium during chronic metabolic acidosis. In the kidney, OGR1 mRNA was found in cells of the glomerulus, proximal tubule, and interstitium including endothelial cells. Wild type (OGR1 &lt;sup&gt;+/+&lt;/sup&gt; ) and OGR1 knockout (OGR1 &lt;sup&gt;-/-&lt;/sup&gt; ) mice were given standard chow without (control) or loaded with ammonium chloride for one or seven days to induce acute or chronic metabolic acidosis, respectively. No differences in responding to the acid load were observed in the knockout mice, except for higher plasma bicarbonate after one day. Bone mineral density, resorption activity of osteoclasts, and urinary deoxypyridinoline were similar between genotypes. During metabolic acidosis the expression levels of key proteins involved in calcium reabsorption, i.e. the sodium/proton exchanger (NHE3), the epithelial calcium-selective channel TRPV5, and the vitamin D-dependent calcium binding protein calbindin-D28k were all higher in the knockout mice compared to wild type mice. This is consistent with the previous demonstration that OGR1 reduces NHE3 activity in proximal tubules of mice. Wild-type mice displayed a non-linear positive association between urinary proton and calcium excretion which was lost in the knockout mice. Thus, OGR1 is a pH sensor involved in the hypercalciuria of metabolic acidosis by controlling NHE3 activity in the proximal tubule. Hence, novel drugs modulating OGR1 activity may improve renal calcium handling