The skeleton provides mechanical support for stature and locomotion, protects vital organs, and controls mineral homeostasis. A healthy skeleton must be maintained by constant bone modeling to carry out these crucial functions throughout life. Bone remodeling involves the removal of old or damaged bone by osteoclasts (bone resorption) and the subsequent replacement of new bone formed by osteoblasts (bone formation). Normal bone remodeling requires a tight coupling of bone resorption to bone formation to guarantee no alteration in bone mass or quality after each remodeling cycle. However, this important physiological process can be derailed by a variety of factors, including menopause-associated hormonal changes, age-related factors, changes in physical activity, drugs, and secondary diseases, which lead to the development of various bone disorders in both women and men. We review the major diseases of bone remodeling, emphasizing our current understanding of the underlying pathophysiological mechanisms.

Background: Multiple laboratory tests are used in the diagnosis and management of patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these assays varies substantially. Approach: An expert committee drafted evidence-based recommendations for the use of laboratory analysis in patients with diabetes. An external panel of experts reviewed a draft of the guidelines, which were modified in response to the reviewers’ suggestions. A revised draft was posted on the Internet and was presented at the AACC Annual Meeting in July, 2000. The recommendations were modified again in response to oral and written comments. The guidelines were reviewed by the Professional Practice Committee of the American Diabetes Association. Content: Measurement of plasma glucose remains the sole diagnostic criterion for diabetes. Monitoring of glycemic control is performed by the patients, who measure their own plasma or blood glucose with meters, and by laboratory analysis of glycated hemoglobin. The potential roles of noninvasive glucose monitoring, genetic testing, autoantibodies, microalbumin, proinsulin, C-peptide, and other analytes are addressed. Summary: The guidelines provide specific recommendations based on published data or derived from expert consensus. Several analytes are of minimal clinical value at the present time, and measurement of them is not recommended.

Oxidative stress plays a critical role in the pathogenesis of atherosclerosis including the formation of lipid laden macrophages and the development of inflammation. However, oxidative stress-induced molecular signaling that regulates the development of vascular calcification has not been investigated in depth. Osteogenic differentiation of vascular smooth muscle cells (VSMC) is critical in the development of calcification in atherosclerotic lesions. An important contributor to oxidative stress in atherosclerotic lesions is the formation of hydrogen peroxide from diverse sources in vascular cells. In this study we defined molecular signaling that is operative in the H 2 O 2 -induced VSMC calcification. We found that H 2 O 2 promotes a phenotypic switch of VSMC from contractile to osteogenic phenotype. This response was associated with an increased expression and transactivity of Runx2, a key transcription factor for osteogenic differentiation. The essential role of Runx2 in oxidative stress-induced VSMC calcification was further confirmed by Runx2 depletion and overexpression. Inhibition of Runx2 using short hairpin RNA blocked VSMC calcification, and adenovirus-mediated overexpression of Runx2 alone induced VSMC calcification. Inhibition of H 2 O 2 -activated AKT signaling blocked VSMC calcification and Runx2 induction concurrently. This blockage did not cause VSMC apoptosis. Taken together, our data demonstrate a critical role for AKT-mediated induction of Runx2 in oxidative stress-induced VSMC calcification.Atherosclerosis is characterized by the presence of atherosclerotic lesions in the arterial intima that leads to narrowing of the vessel lumen. Vascular calcification, the presence of calcium deposits in the vessel wall, is a feature of advanced atherosclerosis and reduces elasticity and compliance of the vessel wall (1). Hence, the extent of calcification is a key risk factor in the pathogenesis of the disease. Several cell types, such as endothelium, monocytes, and vascular smooth muscle cells (VSMC), 5 are involved in different stages of lesion development. VSMC contribute to the development of atherosclerotic lesions through increased migration, proliferation, secretion of matrix components, osteogenic differentiation, and the associated calcification (1). During this process, the differentiated VSMC undergo de-differentiation, and subsequently osteogenic transition that results in vascular calcification (2).Many factors that have been linked to an increased prevalence of vascular calcification are associated with elevated oxidative stress, including hypercholesterolemia, hypertension, diabetes mellitus, and dialysis-dependent end stage renal disease (3-6). Pro-oxidant events in atherosclerosis include the production of reactive oxygen species (ROS) and nitrogen species by vascular cells (7). Of particular interest is hydrogen peroxide (H 2 O 2 ), which is a cell-permeable ROS that has emerged as a key mediator of intracellular signaling (8 -10). H 2 O 2 is produced in vascular cells by multiple enzyma...

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BACKGROUND Multiple laboratory tests are used to diagnose and manage patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these tests varies substantially. APPROACH An expert committee compiled evidence-based recommendations for the use of laboratory testing for patients with diabetes. A new system was developed to grade the overall quality of the evidence and the strength of the recommendations. Draft guidelines were posted on the Internet and presented at the 2007 Arnold O. Beckman Conference. The document was modified in response to oral and written comments, and a revised draft was posted in 2010 and again modified in response to written comments. The National Academy of Clinical Biochemistry and the Evidence Based Laboratory Medicine Committee of the AACC jointly reviewed the guidelines, which were accepted after revisions by the Professional Practice Committee and subsequently approved by the Executive Committee of the American Diabetes Association. CONTENT In addition to long-standing criteria based on measurement of plasma glucose, diabetes can be diagnosed by demonstrating increased blood hemoglobin A1c (Hb A1c) concentrations. Monitoring of glycemic control is performed by self-monitoring of plasma or blood glucose with meters and by laboratory analysis of Hb A1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, proinsulin, C-peptide, and other analytes are addressed. SUMMARY The guidelines provide specific recommendations that are based on published data or derived from expert consensus. Several analytes have minimal clinical value at present, and their measurement is not recommended.

Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogenactivated protein kinases, including extracellular signal-regulated kinase (ERK) and N-terminal Jun kinase (JNK). Here we show, using bovine aortic endothelial cells (BAEC), that shear stress differentially regulates ERK and JNK by mechanisms involving G i2 and pertussis toxin (PTx)-insensitive G-protein-dependent pathways, respectively. Shear activated ERK with a rapid, biphasic time course (maximum by 5 min and basal by 30-min shear exposure) and force dependence (minimum and maximum at 1 and 10 dyn/cm 2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a G i -dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/ cm 2 or higher) with a much slower and prolonged time course (requiring at least 30 min to 4 h) than that of ERK. Also, PTx had no effect on shear-dependent activation of JNK. To further define the shear-sensitive ERK and JNK pathways, vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK were cotransfected with other vectors by using adenoviruspolylysine in BAEC. Expression of the mutant ␣ i2 (G203), antisense G␣ i2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of ␣ i2 (G204) and antisense ␣ i3 did not. Expression of a G␤/␥ scavenger, the carboxyl terminus of ␤-adrenergic receptor kinase (␤ARK-ct), and N17Ras inhibited sheardependent activation of HA-JNK. Treatment of BAEC with genistein prevented shear-dependent activation of ERK and JNK, indicating the essential role of tyrosine kinase(s) in both ERK and JNK pathways. These results provide evidence that 1) G i2 -protein, Ras, and tyrosine kinase(s) are upstream regulators of shear-dependent activation of ERK and 2) that shear-dependent activation of JNK is regulated by mechanisms involving G␤/␥, Ras, and tyrosine kinase(s).Endothelial cells lining the inner vessel wall are in direct contact with flowing blood, which generates a frictional force, hemodynamic shear stress, acting on the surface of the endothelium. Hemodynamic shear stress controls vascular tone, vessel wall remodeling, interaction of blood cells with endothelium, coagulation, and fibrinolysis (1). The focal pattern of atherosclerotic lesions in areas of low and/or unstable shear stress further highlights the importance of shear stress in the atherogenic process (2, 3). Endothelial cells play a key role in shear-dependent vascular changes, sensing shear stress by an unidentified mechanoreceptor(s) followed by production of autocrine and paracrine factors (1). For example, hemodynamic shear stress selectively and differentially regulates production of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, platelet-derived growth factor-B, basic fibroblast growth factor, transforming growth factor ␤-1, tissue plasminogen activator,...

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.

Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity.Introduction: Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects. Materials and Methods: hMSCs were seeded onto plastic microcarrier beads at a density of 10 6 and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM ␤-glycerol phosphate, and 50 M ascorbic acid 2-phosphate. Results: F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88 ± 2% and 77 ± 9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG. Conclusion: Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.