Sarah E. Tague

Sustained Osteomalacia of Long Bones Despite Major Improvement in Other Hypophosphatasia- Related Mineral Deficits in Tissue Nonspecific Alkaline Phosphatase/Nucleotide Pyrophosphatase Phosphodiesterase 1 Double-Deficient Mice

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2-/-) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1-/- mice is corrected in [Akp2-/-; Enpp1-/-] double-knockout mice. Here we have examined the appendicular skeletons of Akp2-/-, Enpp1-/-, and [Akp2-/-; Enpp1-/-] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2-/-, and [Akp2-/-; Enpp1-/-] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2-/- but also in Enpp1-/- and [Akp2-/-; Enpp1-/-] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1-/- calvarial osteoblasts but decreased mineral deposition by Enpp1-/- long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2-/- mice and the hypomineralized phenotype of the long bones of Enpp1-/- mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.

Expression and Synthesis of Bone Morphogenetic Proteins by Osteoclasts: A Possible Path to Anabolic Bone Remodeling

Skeletal remodeling is a finely orchestrated process coupling bone formation to bone resorption. The dynamics of coupling is regulated by the microenvironment at the bone remodeling site, which in turn is influenced by the intercellular communication between cells like osteoclasts and osteoblasts. Understanding the dynamics of coupling is important in devising new therapeutic approaches to the treatment of skeletal diseases characterized by disturbances in the bone remodeling process. In this study, we report the localization of bone morphogenetic proteins (BMPs) in osteoclasts generated from primary cocultures of bone marrow cells from mouse femur and tibia with mouse calvarial osteoblasts, using immunocytochemistry and in situ hybridization. Positive staining was seen in osteoclasts for BMP-2, -4, -6, and -7. Real-time PCR was used to quantitatively confirm the expression of transcripts for BMP-2, BMP-4, and BMP-6 mRNA in murine osteoclasts. Finally, the presence of BMP-2, -4, -6, and-7 proteins was confirmed in osteoclast lysates by Western blotting. Overall, our data suggest a possible direct role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites.

Vitamin D Deficiency Promotes Skeletal Muscle Hypersensitivity and Sensory Hyperinnervation

Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors (“pain-sensing” nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2–4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.

Matrix vesicles are carriers of bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), and noncollagenous matrix proteins

Matrix vesicles (MVs) are well positioned in the growth plate to serve as a carrier of morphogenetic information to nearby chondrocytes and osteoblasts. Bone morphogenetic proteins (BMPs) carried in MVs could promote differentiation of these skeletal cells. Vascular endothelial growth factor (VEGF) in MVs could stimulate angiogenesis. Therefore, a study was undertaken to confirm the presence of bone morphogenetic protein (BMP)-1 through-7, VEGF, and the noncollagenous matrix proteins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) in isolated rat growth plate MVs. MVs were isolated from collagenase-digested rachitic rat tibial and femoral growth plates. The presence of BMP-1 through BMP-7, VEGF, BSP, ON, OPN, and OC was evaluated by Western blot, plus ELISA analyses for BMP-2 and-4 content. The alkaline phosphatase-raising ability of MV extracts on cultured rat growth plate chondrocytes was measured as a reflection of MV ability to promote chondroosseous differentiation. BMP-1 through-7, VEGF, BSP, ON, OPN, and OC were all detected by Western blot analyses. Chondrocytes treated with MV extracts showed a two-to threefold increase in alkaline phosphatase activity over control, indicating increased differentiation. Significant amounts of BMP-2 and BMP-4 were detected in MVs by ELISA. Combined, these data suggest that MVs could play an important morphogenetic role in growth plate and endochondral bone formation.

Vitamin D receptor and enzyme expression in dorsal root ganglia of adult female rats: Modulation by ovarian hormones

Vitamin D insufficiency impacts sensory processes including pain and proprioception, but little is known regarding vitamin D signaling in adult sensory neurons. We analyzed female rat dorsal root ganglia (DRG) for vitamin receptor (VDR) and the vitamin D metabolizing enzymes CYP27B1 and CYP24. Western blots and immunofluorescence revealed the presence of these proteins in sensory neurons. Nuclear VDR immunoreactivity was present within nearly all neurons, while cytoplasmic VDR was found preferentially in unmyelinated calcitonin gene-related peptide (CGRP)-positive neurons, colocalizing with CYP27B1 and CYP24. These data suggest that 1,25(OH)(2)D3 may affect sensory neurons through nuclear or extranuclear signaling pathways. In addition, local vitamin D metabolite concentrations in unmyelinated sensory neurons may be controlled through expression of CYP27B1 and CYP24. Because vitamin D deficiency appears to exacerbate some peri-menopausal pain syndromes, we assessed the effect of ovariectomy on vitamin D-related proteins. Two weeks following ovariectomy, total VDR expression in DRG dropped significantly, owing to a slight decrease in the percentage of total neurons expressing nuclear VDR and a large drop in unmyelinated CGRP-positive neurons expressing cytoplasmic VDR. Total CYP27B1 expression dropped significantly, predominantly due to decreased expression within unmyelinated CGRP-positive neurons. CYP24 expression remained unchanged. Therefore, unmyelinated CGRP-positive neurons appear to have a distinct vitamin D phenotype with hormonally-regulated ligand and receptor levels. These findings imply that vitamin D signaling may play a specialized role in a neural cell population that is primarily nociceptive.

β-adrenoceptor blockers increase cardiac sympathetic innervation by inhibiting autoreceptor suppression of axon growth

β-Adrenoceptor antagonists are used widely to reduce cardiovascular sympathetic tone, but withdrawal is accompanied by sympathetic hyperactivity. Receptor supersensitivity accounts for some but not all aspects of this withdrawal syndrome. Therefore, we investigated effects of β-blockers on sympathetic innervation. Rats received infusions of adrenergic receptor blockers or saline for 1 week. The nonselective β-blocker propranolol and the β1-antagonist metoprolol both increased myocardial sympathetic axon density. At 2 d after propranolol discontinuation, β-receptor sensitivity and responsiveness to isoproterenol were similar to controls. However, tyramine-induced mobilization of norepinephrine stores produced elevated ventricular contractility consistent with enhanced sympathetic neuroeffector properties. In addition, rats undergoing discontinuation showed exaggerated increases in mean arterial pressure in response to air puff or noise startle. In sympathetic neuronal cell cultures, both propranolol and metoprolol increased axon outgrowth but the β2-blocker ICI 118551 did not. Norepinephrine synthesis suppression by α-methyl-p-tyrosine also increased sprouting and concurrent dobutamine administration reduced it, confirming that locally synthesized norepinephrine inhibits outgrowth via β1-adrenoceptors. Immunohistochemistry revealed β1-adrenoceptor protein on sympathetic axon terminations. In rats with coronary artery ligation, propranolol reversed heart failure-induced ventricular myocardial sympathetic axon depletion, but did not affect infarct-associated sympathetic hyperinnervation. We conclude that sympathetic neurons possess β1-autoreceptors that negatively regulate axon outgrowth. Chronic β-adrenoceptor blockade disrupts this feedback system, leading to ventricular sympathetic axon proliferation and increased neuroeffector gain, which are likely to contribute to β-blocker withdrawal syndrome.

Expression of bone morphogenetic proteins and their receptors in the bone marrow megakaryocytes of GATA-1low mice: A possible role in osteosclerosis

The mechanism of osteosclerosis associated with myelofibrosis in megakaryocyte (MK)-related myeloproliferative disorders is largely unknown. However, growth factors released from the bone marrow cells, including from MKs, have been implicated in myelofibrosis, osteosclerosis, and angiogenesis. GATA-1 is a transcription factor required for normal MK development. GATA-1 deficiency in mice (GATA-1low) leads to increased megakaryocytic proliferation, followed by osteosclerosis and myelofibrosis. In this study we investigated the expression of bone morphogenetic proteins (BMPs) and BMP receptors and their possible role in the development of osteosclerosis in the MKs of 12-month-old GATA-1low mice by immunohistochemistry, cytomorphometry, and quantitative real-time PCR. Marrow MKs from both wild-type and GATA-1low mice showed moderate to intense staining for BMP-2, −4, and −6 and BMPR-IA and BMPR-II, whereas splenic MKs showed no BMP immunostaining. Presence of BMP protein in the bone marrow of GATA-1low mice was more than that seen in controls, owing to an increased number of MKs and osteoblasts. The osteosclerosis seen in GATA-1low mice appeared not to be due to a reduced number of functional osteoclasts because the number of tartrate-resistant acid phosphatase-positive osteoclasts was greater in GATA-1low mice than in controls. Our findings demonstrate the presence of significant amounts of BMP-2, −4, and −6 along with their receptors in bone marrow MKs of WT and GATA-1low mice. The increased levels of BMPs appear to be a result of increased numbers of MKs in GATA-1low mice and may, in part, account for the stimulation of osteoblastic activity and resulting osteosclerosis.

Analysis of amino acid neurotransmitters from rat and mouse spinal cords by liquid chromatography with fluorescence detection.

A RP-LC-FL detection method has been developed to identify and quantitate four amino acid neurotransmitters including glutamic acid, glycine, taurine and γ-aminobutyric acid in rat and mouse spinal cord tissue. 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) was employed for the derivatization of these neurotransmitters prior to RP-LC-FL analysis. Different parameters which influenced separation and derivatization were optimized. Under optimum conditions, linearity was achieved within the concentration ranges of 0.50-50.00 μM for all analytes with correlation coefficients from 0.9912 to 0.9997. The LODs ranged from 0.03 μM to 0.06 μM. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as rat and mouse spinal cord with satisfactory recoveries.

Vitamin D deficiency leads to sensory and sympathetic denervation of the rat synovium.

Vitamin D deficiency is associated with increased susceptibility to inflammatory arthritis. Sensory and sympathetic synovial nerves are critical to the development of inflammatory arthritis and spontaneously degenerate in the early phases of disease. These nerves contain vitamin D receptors and vitamin D influences nerve growth and neurotrophin expression. We therefore examined the density of synovial nerves and neurotrophin-containing cells in vitamin D-deficient rats. Seven-week-old Sprague-Dawley rats were fed either control or vitamin D-deficient diets for 4weeks. Knee synovium sections extending from the patella to the meniscus were immunostained for total nerves, myelinated and unmyelinated nerves, sympathetic nerves, peptidergic and non-peptidergic sensory nerves, and neurotrophins and immune cell markers. In control rats, intimal innervation by unmyelinated sensory fibers was denser than subintimal innervation. In contrast, sympathetic innervation was confined to the subintima. Many sensory axons contained markers for both peptidergic and non-peptidergic nerves. Nerve growth factor (NGF) was primarily expressed by intimal CD163-negative type B synoviocytes, while neurturin, a ligand selective for non-peptidergic sensory neurons, was expressed by synovial mast cells. In vitamin D-deficient rats, there were significant reductions in sensory nerves in the intima and sympathetic nerves in the subintima. While there was no significant change in NGF-immunoreactivity, the number of neurturin-expressing mast cells was significantly reduced in the intima, suggesting that intimal reductions in sensory nerves may be related to reductions in neurturin. Vitamin D deficiency therefore may increase susceptibility to inflammatory arthritis by depleting sensory and sympathetic synovial nerves as a result of reduced synovial neurotrophin content.