Elamipretide mitigates ischemia-reperfusion injury in a swine model of hemorrhagic shock.

ischemia-reperfusion injury (IRI) after hemorrhage is potentiated by aortic occlusion or resuscitative endovascular balloon occlusion of the aorta (REBOA). Given the central role of mitochondrial injury in shock, we hypothesized that Elamipretide, a peptide that protects mitochondria, would mitigate IRI after hemorrhagic shock and REBOA. Twelve pigs were subjected to hemorrhagic shock and 45 min of REBOA. After 25 min of REBOA, animals received either saline or Elamipretide. Animals were transfused with autologous blood during balloon deflation, and pigs were resuscitated with isotonic crystalloids and norepinephrine for 4.25 h. Elamipretide-treated animals required less crystalloids than the controls (62.5 [50-90] and 25 [5-30] mL/kg, respectively), but similar amounts of norepinephrine (24.7 [8.6-39.3] and 9.7 [2.1-12.5] mcg/kg, respectively). Treatment animals had a significant reduction in serum creatinine (control: 2.7 [2.6-2.8]; Elamipretide: 2.4 [2.4-2.5] mg/dL; p = 0.04), troponin (control: 3.20 [2.14-5.47] ng/mL, Elamipretide: 0.22 [0.1-1.91] ng/mL; p = 0.03), and interleukin-6 concentrations at the end of the study. There were no differences in final plasma lactate concentration. Elamipretide reduced fluid requirements and protected the kidney and heart after profound IRI. Further understanding the subcellular consequences of REBOA and mitochondrial rescue will open new therapeutic avenues for patients suffering from IRI after hemorrhage.

Network meta-analysis of lorcaserin and oral hypoglycaemics for patients with type 2 diabetes mellitus and obesity.

In addition to weight loss, randomized controlled trials have shown improvement in glycaemic control in patients taking lorcaserin. The aim of this study aim was to compare adding lorcaserin or other glucose lowering medications to metformin on weight and glycaemic control. A systematic review and network meta-analysis of randomized controlled trials were conducted. Included studies (published 1990-2014) were of lorcaserin or glucose lowering medications in type 2 diabetic patients compared to placebo or different active treatments. Studies had to report ≥1 key outcome (change in weight or HbA1c, % HbA1c <7, hypoglycaemia). Direct meta-analysis was performed using DerSimonian and Laird random effects models, and network meta-analysis with Bayesian Markov-chain Monte Carlo random effects models; 6552 articles were screened and 41 included. Lorcaserin reduced weight significantly more than thiazolidinediones, glinides, sulphonylureas and dipeptidyl peptidase-4 inhibitors, some of which may have led to weight gain. There were no significant differences in weight change between lorcaserin and alpha-glucoside inhibitors, glucagon-like peptide-1 agonists and sodium/glucose cotransporter 2 inhibitors. Network meta-analysis showed lorcaserin was non-inferior to all other agents on HbA1c reduction and % achieving HbA1c of <7%. The risk of hypoglycaemia was not significantly different among studied agents except that sulphonylureas were associated with higher risk of hypoglycaemia than lorcaserin. Although additional studies are needed, this analysis suggests in a population of patients with a body mas index of ≥27 who do not achieve glycaemic control on a single agent, lorcaserin may be added as an alternative to an add-on glucose lowering medication.

Calcitonin induces dephosphorylation of Pyk2 and phosphorylation of focal adhesion kinase in osteoclasts.

Calcitonin induces the association and tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and HEF1 in HEK-293 cells that overexpress the calcitonin receptor (C1a-HEK), but the hormone's effect on these adhesion-related proteins in osteoclasts is not known. We therefore studied the effect of calcitonin on the tyrosine phosphorylation and subcellular distribution of paxillin, HEF1, FAK, and Pyk2, a FAK-related tyrosine kinase, in osteoclasts. Osteoclasts expressed both Pyk2 and FAK, with Pyk2 much more highly expressed. The two tyrosine kinases and paxillin were prominently associated with small punctate structures that were most densely clustered in the region of the peripheral F-actin-rich ring. Some of the punctate structures stained either for Pyk2 alone or FAK alone. Treatment with calcitonin disrupted the actin ring and induced the loss of the peripheral staining of paxillin, Pyk2, and FAK. In calcitonin-treated osteoclast-like cells, the tyrosine phosphorylation of paxillin and FAK increased, whereas the tyrosine phosphorylation of Pyk2 decreased. Calcitonin also induced increased phosphorylation of Erk1 and Erk2 in osteoclasts, as it did in the C1a-HEK cells. The unexpected dephosphorylation of Pyk2 correlated with decreased phosphorylation of Tyr(402), the autophosphorylation site of Pyk2. The calcitonin-induced dephosphorylation of Pyk2 was not observed in C1a-HEK cells transfected with Pyk2, suggesting that the reduced phosphorylation seen in osteoclasts may be specific to these cells. Treatment of osteoclast-like cells with 12-phorbol 13-myristate acetate increased the tyrosine phosphorylation of both Pyk2 and FAK, and calphostin C, an inhibitor of protein kinase C, blocked calcitonin-stimulated FAK phosphorylation. Increasing intracellular calcium with ionomycin caused a decrease in the tyrosine phosphorylation of Pyk2 and the loss of the actin ring in a manner similar to the effect of calcitonin. Ionomycin had no effect on FAK tyrosine phosphorylation. Calcitonin (CT)-induced changes in Pyk2, FAK, and Erk1/2 phosphorylation were independent of c-Src.

Omission of active commuting to school and the prevalence of children's health-related physical activity levels: the Russian Longitudinal Monitoring Study.

BACKGROUND: Active commuting to school by walking or bicycle is a potential source of continuous moderate activity for children that has been largely ignored in surveys of physical activity. The purpose of this study was to evaluate the analytical impact of omitting active commuting to school (walking or bicycling) on conclusions about children's physical activity levels. METHODS: The Russian Longitudinal Monitoring Study (RLMS) is the first nationally representative household survey in the Russian Federation. More than 6400 households from all regions of Russia were surveyed eight times between 1992 and 1998. Analysis was conducted using physical activity data (school physical education classes, out-of-school active pursuits and active commuting to school) obtained by parent-proxy on 1094 (572 boys, 522 girls) school-aged Russian children (mean age 10.2 +/- 1.9 years) participating in the November 1998 round of the RLMS. Data were examined according to prevalence of achievement of health-related physical activity guidelines, active commuting to school behaviours included then omitted. RESULTS: Omitting active commuting to school resulted in a statistically significant decrease in the prevalence of achievement of health-related guidelines from 12% to 20%, similar for both genders. Likewise, the prevalence of sedentarism (defined as not meeting any of the guidelines) was increased by 17-22%. CONCLUSIONS: The present findings suggest that, in order to avoid misclassification bias of children's physical activity levels, it is necessary to include questions about mode of commuting to school. The findings also carry practice implications: the commonplace need to get to and from school may be a missed opportunity for children's health-related physical activity in motorized societies.

Limitations on the use of a single screening question to measure sedentary behavior.

OBJECTIVES: This study explored the limitations of identifying sedentary individuals via an existing screening question in a state-based surveillance system. METHODS: A national sample (n = 7529) of adults, selected by random-digit dialing between November 1999 and May 2000, responded about participation in leisure-time physical activity. RESULTS: Of those who initially reported no leisure-time physical activity (25%), 85% were engaging in at least some activity, and 20% were engaging in enough moderate- or vigorous-intensity activity to meet health-related recommendations. CONCLUSIONS: Public health programs that use only 1 screening question to identify sedentary behavior may not be able to target physical activity messages effectively, especially if physical activity is defined to include a broad range of activities beyond sports.

NF-kappaB and the MAP kinases/AP-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein I/II, a modulin from oral streptococci.

As in rheumatoid arthritis (RA), it was demonstrated recently that bacterial fragments of DNA or rRNA are present in the joint and therefore could play a role in inducing or perpetuating the disease, this work was initiated to define mechanisms that account for the stimulatory activities of the oral streptococcal modulin, protein I/II, on fibroblast-like synoviocytes (FLSs) from RA patients. FLSs from RA patients were stimulated with protein I/II, and expression of interleukin (IL)-6 and IL-8 mRNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Immunoblotting by antibodies specific for activated forms of MAPKs and electrophoretic mobility shift assays (EMSAs) were performed to study downstream signalling, which allowed the synthesis of IL-6 and IL-8. We reported that protein I/II interactions with FLSs from RA patients trigger the synthesis and release of IL-6 and IL-8. We also demonstrated that protein I/II enhances the phosphorylation of ERK 1/2, p38 and JNKs and that ERK 1/2 and JNK MAPKs seem to play a more important role than p38 in protein I/II-mediated synthesis of IL-6 and IL-8. Our experiments also indicated that stimulation of FLSs with protein I/II induces nuclear translocation of NF-kappaB, AP-1-binding activity and that NF-kappaB plays a major role in IL-6 and IL-8 secretion from activated cells.

The mouse osteopetrotic grey-lethal mutation induces a defect in osteoclast maturation/function.

The osteopetrotic grey-lethal (gl) mouse mutant displays many similarities to the human malignant autosomal-recessive form of osteopetrosis. In this study, we show that the gl osteopetrotic bone phenotype is characterized by the presence of numerous differentiated multinucleated osteoclasts. A significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was detected in vivo, suggesting induction of differentiation in the osteoclast lineage as a compensatory mechanism. These gl osteoclast cells demonstrated a defective cytoskeletal reorganization and an underdeveloped ruffled border, a membrane structure essential for active bone resorption. Accordingly, resorption activity of these cells is markedly impaired by four- to tenfold as evaluated with the pit formation assay. This low bone resorption in gl osteoclasts is highly reminiscent of the loss in key enzymes, V-ATPase or cathepsin-K, and in signaling factors, Src or TRAF-6, which were shown not to be significantly altered in gl osteoclasts. Thus, independently of a deficiency in V-ATPase, Src, cathepsin-K, and TRAF-6, the gl mutation results in increased number of osteoclasts, characterized by a disrupted cytoskeleton and an underdeveloped ruffled border.

Carbonic anhydrase II is an AP-1 target gene in osteoclasts.

c-Fos, a member of the AP-1 family of transcription factors, is necessary for osteoclast differentiation but to date, none of the osteoclast-phenotypic markers have been identified as AP-1 target genes. Here, we demonstrate that carbonic anhydrase II (CA II), an enzyme necessary for osteoclast activity, is transcriptionally upregulated by c-Fos/AP-1. A functional AP-1 binding site is present in the CA II promoter and is necessary for this regulation. Furthermore, we show that AP-1 binding activity, mainly composed of Fra-2 and JunD, is induced by treatment of bone marrow cultures with the osteoclastogenic hormone 1,25 dihydroxyvitamin D(3). Fra-2 and JunD are found in mature osteoclasts as well. Thus, our data demonstrate that cFos/AP-1 can directly regulate the expression of this osteoclast marker and that AP-1 activity is upregulated in osteoclast progenitors in response to osteoclastogenic signals.

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility.

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.

Progressive increase in bone mass and development of odontomas in aging osteopetrotic c-src-deficient mice.

The critical role of c-src in osteoclast-mediated bone resorption has been emphasized by gene deletion experiments in mice. However, the long-term effects of the lack of c-src and impaired osteoclast function on the skeleton remain unknown. To further study the physiological role of c-src and to circumvent the early death of src(-/-) mice, due to starvation in the absence of erupted teeth, we maintained mice on a liquid diet. At the age of 2 months the src(-/-) mice presented signs of airway obstruction and all mice died progressively between 2.5 and 6 months of age. Radiography demonstrated severe osteopetrosis of the whole skeleton. Histomorphometrical analysis of the src(-/-) mice confirmed a significant increase in bone mass with age, resulting in complete loss of bone marrow spaces in some bones and explaining the consistent hepatosplenomegaly, due to extraskeletal hematopoesis. Histopathological examination of the skull revealed the presence of odontomas in the region of the unerupted incisors, with a penetrance of 100% in the aging src(-/-) mice. Although odontomas are benign lesions, their progressive growth leads to the obliteration of the nasal airways, progressive suffocation, and death in src(-/-) mice. These results suggest that: (i) in the absence of bone resorption, bone formation continues and leads to progressive accentuation of the osteopetrotic phenotype in src(-/-) mice; (ii) osteoclastic function is required for regular eruption of the incisors and deficient bone resorption is associated with the development of odontomas; and (iii) src(-/-) mice die by suffocation due to airway obliteration as a result of progressive odontoma growth.

Physical activity surveillance in the 21(st) century.

PURPOSE: The 1996 Surgeon General's Report on Physical Activity and Health emphasized the health-related benefits of moderate-intensity physical activities, especially everyday activities. Unfortunately most surveillance systems in the U.S. primarily measure sports-related activities, particularly vigorous intensity sports. This report describes a new physical activity surveillance instrument designed to go beyond our current measures and include moderate-intensity everyday activities.METHODS: Data were collected from a nationally representative sample of adults (n = 5010). Questions were asked about occupational activity (mostly sitting; mostly walking; mostly heavy labor); walking (for exercise, transportation, or any other reason); moderate-intensity activities (brisk walking, yard work, vacuuming); vigorous-intensity activities (running, aerobics, heavy yard work); and strengthening activities (lifting weights, pull-ups, sit-ups). Questions included frequency and duration of activities. Respondents were defined as recommended if they participated in either moderate-intensity activities >/=30 min/day for >/=5 days/wk OR vigorous-intensity activities >/=20 min/day for >/=3 days/wk. Insufficient was defined as not meeting recommended levels while inactive was defined as no leisure-time activity.RESULTS: Overall 40% of adults were in the recommended group, 44% were in the insufficient group and 16% were inactive. Among working adults 37% had jobs that involved mostly walking or heavy labor and about 50% of those also were in the recommended group. Among the 63% of working adults who report mostly sitting at work, 39% were in the recommended group.CONCLUSIONS: These results suggest that measuring only leisure-time physical activity may under represent the physical activity experience of many U.S. adults. Broadening the concept of physical activity beyond traditional sports-related vigorous "exercise" may provide a more accurate picture of the prevalence of health-related physical activity.

Spinal cholinergic inhibition of the pressor response to muscle activation is mediated by muscarinic, but not nicotinic, receptors.

This study examined the influence of spinal muscarinic and nicotinic receptors on the cardiovascular adjustments to skeletal muscle activation in anesthetized cats. Microdialyzing into the L(7) dorsal horn increasing doses of the muscarinic receptor agonist bethanechol, but not the nicotinic receptor antagonist mecamylamine, reduced increases in mean arterial pressure (MAP) and heart rate (HR) during hindlimb contraction or passive stretch. Atropine administration accentuated the cardiovascular responses during contraction, but not during passive stretch. These data indicate that muscarinic, but not nicotinic, receptors at the dorsal horn level blunt the pressor response to muscle activity. Further, the data suggest that the two neural pathways involved in muscle contraction or stretch are anatomically distinct.

A functional link between dynamin and the actin cytoskeleton at podosomes.

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

Proteinases in bone resorption: obvious and less obvious roles.

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

The tyrosine phosphatase SHP-1 is a negative regulator of osteoclastogenesis and osteoclast resorbing activity: increased resorption and osteopenia in me(v)/me(v) mutant mice.

Naturally occuring inactivating mutations of the Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 (SHP-1) in mice give rise to the motheaten (me) phenotype. me/me mice have multiple hematopoietic abnormalities, suggesting that this phosphatase plays an important role in hematopoiesis. SHP-1 binds to and is activated by several hematopoietic surface receptors, including the colony-stimulating factor type 1 receptor. We have examined the role of SHP-1 in osteoclastogenesis and osteoclast function using mice with the viable motheaten (me(v)/me(v)) mutation, which has markedly decreased SHP-1 activity. Histomorphometric analysis of 6-week-old me(v)/me(v) mice and control littermates showed a marked osteopenia with an increase in bone resorption indices. The number of formed osteoclast-like cells (OCLs) in cocultures of me(v)/me(v) hematopoietic cells with normal osteoblasts was significantly increased. In contrast, the number of OCLs formed in the coculture of normal bone marrow cells with the me(v)/me(v) osteoblasts was not significantly different from controls. The bone-resorbing activity of me(v)me(v) OCLs and authentic osteoclasts was also found to be increased. Finally, Western blotting of proteins from me(v)/me(v) and control OCLs revealed an overall increase in tyrosine phosphorylation in the me(v)/me(v) lysates. These in vivo and in vitro results suggest that SHP-1 is a negative regulator of bone resorption, affecting both the formation and the function of osteoclasts.

Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind to and activate the same PTH/PTHrP receptor. Deletion of either the PTHrP gene or the PTH/PTHrP receptor gene leads to acceleration of differentiation of growth plate chondrocytes. To explore further the functional relationships of PTHrP and the PTH/PTHrP receptor, bones of knockout mice were analyzed early in development, and the phenotypes of double-knockout mice were characterized. One early phenotype is shared by both knockouts. Normally, the first chondrocytes to become hypertrophic are located in the centers of long bones; this polarity is greatly diminished in both these knockouts. The PTH/PTHrP receptor-deficient (PTH/PTHrP-R(-/-)) mice exhibited 2 unique phenotypes not shared by the PTHrP(-/-) mice. During intramembranous bone formation in the shafts of long bones, only the PTH/PTHrP-R(-/-) bones exhibit a striking increase in osteoblast number and matrix accumulation. Furthermore, the PTH/PTHrP-R(-/-) mice showed a dramatic decrease in trabecular bone formation in the primary spongiosa and a delay in vascular invasion of the early cartilage model. In the double-homozygous knockout mice, the delay in vascular invasion did not occur. Thus, PTHrP must slow vascular invasion by a mechanism independent of the PTH/PTHrP receptor.

Thy-1 antigen expression by cells in the osteoblast lineage.

Identification of surface markers involved in osteoblast differentiation provides a method to isolate osteoblasts at various stages of maturation. In this study, we examined expression of the T lymphocyte differentiation antigen, Thy-1, by osteoblastic cells from different species. Murine skeletal progenitor, neonatal calvarial, and adult bone cells (ABCs) were selected to represent osteoblasts at distinct stages of maturation. Flow cytometric analysis showed that Thy-1 expression was undetectable on the progenitor cells (mouse limb bud clones 14 and 17), appeared on calvarial cells (45%+), and was decreased on ABCs (< 10%+). Thy-1 was also detected in situ on osteoblastic cells in mouse calvariae. Thy-1 mRNA expression correlated with cell surface expression. Antigen expression was markedly increased during the cells' proliferative phase in culture. Furthermore, examination of primary rat and human osteoblast-like cells revealed that significant levels of Thy-1 were also expressed on those cells derived from subconfluent culture. This study indicates that osteoblasts express Thy-1 antigen and that its expression is maximal at their earliest stage of maturation, during the proliferative phase, and then declines as the cells mature. In a role similar to the one it plays in the hematopoietic system, Thy-1 antigen may be useful as a differentiation marker in following the development of the osteoblast.