Andreas Niemeier

Brain Gray Matter Decrease in Chronic Pain Is the Consequence and Not the Cause of Pain

Recently, local morphologic alterations of the brain in areas ascribable to the transmission of pain were reported in patients suffering from chronic pain. Although some authors discussed these findings as damage or loss of brain gray matter, one of the key questions is whether these structural alterations in the cerebral pain-transmitting network precede or succeed the chronicity of pain. We investigated 32 patients with chronic pain due to primary hip osteoarthritis and found a characteristic gray matter decrease in patients compared with controls in the anterior cingulate cortex (ACC), right insular cortex and operculum, dorsolateral prefrontal cortex (DLPFC), amygdala, and brainstem. We then investigated a subgroup of these patients (n = 10) 6 weeks and 4 months after total hip replacement surgery, monitoring whole brain structure. After surgery, all 10 patients were completely pain free and we observed a gray matter increase in the DLPFC, ACC, amygdala, and brainstem. As gray matter decrease is at least partly reversible when pain is successfully treated, we suggest that the gray matter abnormalities found in chronic pain do not reflect brain damage but rather are a reversible consequence of chronic nociceptive transmission, which normalizes when the pain is adequately treated.

Lrp4, a novel receptor for dickkopf 1 and sclerostin, is expressed by osteoblasts and regulates bone growth and turnover In Vivo

Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1) is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC) and bone mineral density (BMD). Lumbar spine trabecular bone volume per total volume (BV/TV) was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways.

Endothelial-derived lipoprotein lipase is bound to postprandial triglyceride-rich lipoproteins and mediates their hepatic clearance in vivo.

Abstract. Lipoprotein lipase (LPL) is the key enzyme in the intravascular hydrolysis of triglyceride-rich lipoproteins (TRL). Furthermore, it has been shown that inactive LPL can mediate cellular binding and uptake of TRL in vitro. This study investigated whether LPL is bound to postprandial human TRL in vivo, and whether it plays a role in the hepatic clearance of these particles independent of its catalytic activity. LPL was found to bind to postprandial TRL in preheparin plasma of healthy young men. To study the effect of inactive LPL on particle uptake, TRL isolated from patients with inactive LPL (LPL or apoC-II mutations) were used before and after heparin administration. These model particles allow one to study the bridging effect of LPL independent of its enzymatic activity. Organ uptake studies with these particles in mice revealed that inactive LPL increases the hepatic clearance of TRL significantly while uptake into other organs remains largely unaffected. Further evidence that endothelial-derived LPL directs TRL to the liver in vivo was gained with transgenic mice that express inactive LPL exclusively in muscle, revealing greater hepatic uptake than in wild-type mice. In conclusion, these data demonstrate for the first time that LPL is a structural component of postprandial TRL which facilitates hepatic TRL clearance from the circulation independent of its catalytic function.

Increased Bone Formation in Mice Lacking Apolipoprotein E

ApoE is a plasma protein that plays a major role in lipoprotein metabolism. Here we describe that ApoE expression is strongly induced on mineralization of primary osteoblast cultures. ApoE‐deficient mice display an increased bone formation rate compared with wildtype controls, thereby showing that ApoE has a physiologic function in bone remodeling.

Signature of circulating microRNAs in osteoarthritis

BACKGROUND Osteoarthritis is the most common form of arthritis and a major socioeconomic burden. Our study is the first to explore the association between serum microRNA levels and the development of severe osteoarthritis of the knee and hip joint in the general population. METHODS We followed 816 Caucasian individuals from 1995 to 2010 and assessed joint arthroplasty as a definitive outcome of severe osteoarthritis of the knee and hip. After a microarray screen, we validated 12 microRNAs by real-time PCR in the entire cohort at baseline. RESULTS In Cox regression analysis, three microRNAs were associated with severe knee and hip osteoarthritis. let-7e was a negative predictor for total joint arthroplasty with an adjusted HR of 0.75 (95% CI 0.58 to 0.96; p=0.021) when normalised to U6, and 0.76 (95% CI 0.6 to 0.97; p=0.026) after normalisation to the Ct average. miRNA-454 was inversely correlated with severe knee or hip osteoarthritis with an adjusted HR of 0.77 (95% CI 0.61 to 0.97; p=0.028) when normalised to U6. This correlation was lost when data were normalised to Ct average (p=0.118). Finally, miRNA-885-5p showed a trend towards a positive relationship with arthroplasty when normalised to U6 (HR 1.24; 95% CI 0.95 to 1.62; p=0.107) or to Ct average (HR 1.30; 95% CI 0.99 to 1.70; p=0.056). CONCLUSIONS Our study is the first to identify differentially expressed circulating microRNAs in osteoarthritis patients necessitating arthroplasty in a large, population-based cohort. Among these microRNAs, let-7e emerged as potential predictor for severe knee or hip osteoarthritis.

Expression of LRP1 by Human Osteoblasts: A Mechanism for the Delivery of Lipoproteins and Vitamin K1 to Bone

Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts.

Structural Brain Changes in Chronic Pain Reflect Probably Neither Damage Nor Atrophy

Chronic pain appears to be associated with brain gray matter reduction in areas ascribable to the transmission of pain. The morphological processes underlying these structural changes, probably following functional reorganisation and central plasticity in the brain, remain unclear. The pain in hip osteoarthritis is one of the few chronic pain syndromes which are principally curable. We investigated 20 patients with chronic pain due to unilateral coxarthrosis (mean age 63.25±9.46 (SD) years, 10 female) before hip joint endoprosthetic surgery (pain state) and monitored brain structural changes up to 1 year after surgery: 6–8 weeks, 12–18 weeks and 10–14 month when completely pain free. Patients with chronic pain due to unilateral coxarthrosis had significantly less gray matter compared to controls in the anterior cingulate cortex (ACC), insular cortex and operculum, dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex. These regions function as multi-integrative structures during the experience and the anticipation of pain. When the patients were pain free after recovery from endoprosthetic surgery, a gray matter increase in nearly the same areas was found. We also found a progressive increase of brain gray matter in the premotor cortex and the supplementary motor area (SMA). We conclude that gray matter abnormalities in chronic pain are not the cause, but secondary to the disease and are at least in part due to changes in motor function and bodily integration.

Identification of Megalin/gp330 as a Receptor for Lipoprotein(a) In Vitro

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein of unknown physiological function. The mechanism of Lp(a) atherogenicity as well as its catabolic pathways are only incompletely understood at present. In this report, we show that the low density lipoprotein receptor (LDLR) gene family member megalin/glycoprotein (gp) 330 is capable of binding and mediating the cellular uptake and degradation of Lp(a) in vitro. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient in LDLR-related protein (LRP) and a control cell line carrying a double knockout for both LRP and megalin/gp330 were compared with regard to their ability to bind, internalize, and degrade dioctadecyltetramethylindocarbocyanine perchlorate (DiI)-fluorescence-labeled Lp(a) as well as equimolar amounts of 125I-labeled Lp(a) and LDL. Uptake and degradation of radiolabeled Lp(a) by the megalin/gp330-expressing cells were, on average, 2-fold higher than that of control cells. This difference could be completely abolished by addition of the receptor-associated protein, an inhibitor of ligand binding to megalin/gp330. Mutual suppression of the uptake of 125I-Lp(a) and of 125I-LDL by both unlabeled Lp(a) and LDL suggested that Lp(a) uptake is mediated at least partially by apolipoprotein B100. Binding and uptake of DiI-Lp(a) resulted in strong signals on megalin/gp330-expressing cells versus background only on control cells. In addition, we show that purified megalin/gp330, immobilized on a sensor chip, directly binds Lp(a) in a Ca2+-dependent manner with an affinity similar to that for LDL. We conclude that megalin/gp330 binds Lp(a) in vitro and is capable of mediating its cellular uptake and degradation.

Uptake of postprandial lipoproteins into bone in vivo: Impact on osteoblast function

Dietary lipids and lipophilic vitamins are transported by postprandial lipoproteins and are required for bone metabolism. Despite that, it remains unknown whether bone cells are involved in the uptake of circulating postprandial lipoproteins in vivo. The current study was performed to investigate a putative participation of bone in the systemic postprandial lipoprotein metabolism in mice, to identify potentially involved cell type populations and to analyze whether lipoprotein uptake affects bone function in vivo. As a model for the postprandial state, chylomicron remnants (CR) were injected intravenously into mice. Next to the liver and compared to other organs, bone appeared to be the second most important organ for the clearance of radiolabeled CR particles from the circulation in vivo. In addition, uptake of radiolabeled CR by primary murine osteoblasts and hepatocytes was quantified to be in a similar range in vitro. A complementary approach with fluorescently labeled CR and immunohistochemical staining for apoE proved that intact CR particles were taken up into bone and liver. Electron microscopy localization studies of bone sections revealed CR uptake into sinusoidal endothelial cells, macrophages and osteoblasts. The relative amount of radiolabeled CR uptake into femoral cortical bone, representing predominantly osteoblasts, and bone marrow, representing predominantly non-osteoblast cells, was within the same range. Most importantly, the injection of vitamin K1-enriched CR resulted in an increase of the degree of osteocalcin carboxylation in vivo while total osteocalcin concentrations remained unaffected, giving functional proof that osteoblasts process CR in vivo. In conclusion, here we demonstrate that bone is involved in the postprandial lipoprotein metabolism in mice. Osteoblasts participate in CR clearance from the circulation, which has a direct impact on the secretory function of osteoblasts.

Knockdown of Hepatic ABCA1 by RNA Interference Decreases Plasma HDL Cholesterol Levels and Influences Postprandial Lipemia in Mice

Objective—To investigate the impact of hepatic ABCA1 on systemic lipoprotein metabolism in vivo by an adenovirus-mediated RNA interference approach. Methods and Results—Efficiency of plasmid-based small interference RNA (siRNA)-induced knockdown of cotransfected murine ATP binding cassette transporter A1 (mABCA1) in HEK-293 cells was judged by RT-polymerase chain reaction, immunofluorescence, and Western blot analysis. The most effective plasmid was used to generate a recombinant adenovirus as a tool to selectively downregulate ABCA1 expression in mouse liver (C57BL/6). In comparison to controls, Western blot analysis from liver membrane proteins of Ad-anti-ABCA1 infected mice resulted in an ≈50% reduction of endogenous ABCA1 and a clear upregulation of apolipoprotein E. Fast protein liquid chromatography analysis of plasma revealed that hepatic ABCA1 protein reduction was associated with an ≈40% decrease of HDL cholesterol and a reduction of HDL-associated apolipoprotein A-I and E. In the fasted state, other lipoprotein classes were not affected. To analyze the influence of ABCA1 downregulation on postprandial lipemia, infected mice were given a gastric load of radiolabeled trioleate in olive oil. In Ad-anti-ABCA1 infected mice, the postprandial increase of chylomicrons and chylomicron-associated apolipoproteins B and E was significantly reduced as compared with controls. Conclusion—Hepatic ABCA1 contributes to HDL plasma levels and influences postprandial lipemia.