Adrenocorticotropin Hormone Expression in the Developing Chicken Limb

In previous studies using mammalian models we have found both clinical and laboratory evidence of a role for melanocortins in endochondral ossification. The melanocortin system has remarkable conservation among vertebrates and melanocortin receptors are expressed with significant sequence homology in teleosts to mammals. The overall goal of these studies is to provide a more accessible model of melanocortin involvement in endochondral growth. We hope to determine if melanocortins play a role during endochondral ossification of the developing chicken limb. Like in mammals melanocortins are widely distributed throughout the body of chicken and participate in a wide range of physiological functions with the peripheral tissue distribution of melanocortin receptors in chicken more widespread. Our first step was to examine melanocortin expression in the developing limbs of the chick embryo. Using immunohistochemistry techniques, we detected ACTH (1-24) in the limbs of embryonic day 9 chick embryos. This initial data indicates that the chick embryo is a viable model that can be used to determine a role for melanocortin in endochondral growth. Melanocortin expression shows remarkable sequence homology, therefore results of these studies can be extrapolated to many vertebrate models

ACTH Enhances Lipid Accumulation in Bone-marrow derived Mesenchymal stem cells undergoing adipogenesis

ACTH is a major hormone of the stress axis or hypothalamic-pituitary-adrenal (HPA) axis. It is derived from pro-opiomelanocortin (POMC) the precursor to the melanocortin family of peptides. POMC produces the biologically active melanocortin peptides via a series of enzymatic steps in a tissue-specific manner, yielding the melanocyte-stimulating hormones (MSHs), corticotrophin (ACTH) and β-endorphin. The melanocortin system plays an imperative role in energy expenditure, insulin release and insulin sensitivity. Bone marrow derived mesenchymal stem cells circulate in the blood stream and as progenitor cells have the potential to differentiate into many cell types such as osteoblasts, chondrocytes and adipocytes. Here we examine the effects of ACTH on the mouse D1 bone marrow-derived MSC. ACTH significantly increased lipid accumulation during the adipogenic differentiation of D1 cells in a concentration- dependent manner. ACTH also shifts the temporal pattern of D1 adipogenic differentiation to the left i.e. differentiation occurs earlier with ACTH treatment. No significant differences in protein expression of peroxisome proliferator-activated receptor gamma (PPAR-γ2), a regulating transcription factor of adipogenesis were found. Therefore the effects of ACTH are suggested to be mediated by an alternative pathway. Overall the results indicate a connection between increased adipose deposition and the elevated circulating ACTH associated with stress

Stress Axis Hormones Induce Triglyceride Filled Nodule Formation in Vascular Smooth Muscle Cells

Homeostatic stress, such as that which occurs in diabetes, is associated with increased risk for the development of atherosclerosis. Atherosclerotic plaques of the artery wall are associated with both lipid accumulation and fibrous and/or calcified tissue accumulation. Vascular smooth muscle cells (VSMC) are derived from mesenchymal stem cells (MSC) which are capable of differentiating into adipocytes, chondrocytes and osteoblasts. MSC of the bone marrow are pushed toward the chondrogenic and adipogenic phenotypes in the presence of the stress hormones glucocorticoid and adrenocorticotropin (ACTH). This led us to hypothesize that the proliferative VSMC of the Goto-Kakizaki (GK) diabetic rat, when exposed to stress hormones will present an adipocytic and/or chondrogenic-like phenotype. VSMC of the GK rat were cultured using conditions that favor the multi-potential differentiation of MSC and were either left untreated, were treated with ACTH, dexamethasone (DEXA) or both. Cells were stained for lipid using oil-red-o, proteoglycan matrix using alcian blue and cell density using methylene blue. DEXA increased lipid nodule formation above the untreated control but the combined ACTH and DEXA treatment led to a significant increase above DEXA alone (lipid nodule #’s per field, DEXA 2.56 ± 1.63 vs. A+D 6.67 ± 1.68). These data suggest that stress hormones may contribute to VSMC matrix accumulation and lipid production during atherosclerosis development in diabetes

High Glucose Enhances the Proliferation Effects of Stress Hormones in Mesenchymal Stem Cell Cultures

Mesenchymal stem cells (MSC) are the progenitor cells to connective tissue cells, epithelial cells, and smooth muscle cells. These cells are currently being investigated as a cellular source for tissue regeneration and repair. The systemic and local tissue environment may have significant influence on the success of such efforts. We hypothesized that elevated glucose and exposure to stress hormones would influence the proliferation of MSC. MSC from the bone marrow of Wistar-Kyoto (WKY) rats were grown under both low glucose (5 mM) and high glucose (20 nM) conditions in the presence and absence of the synthetic glucocorticoid, dexamethasone, and adrenocorticotropin hormone (ACTH). Relative cell density was used to determine the rate of proliferation and was measured using methylene blue staining. Changes in cell density from initial plating were recorded at various stages of culture. Under low-glucose conditions, stress hormones reduced MSC proliferation and these changes were enhanced when cells were exposed to high glucose conditions. These data indicate that stress and elevated glucose can have a significant effect on the ability of MSC to proliferate and may prove to reduce their efficacy during tissue repair

ACTH Promotes Osteogenesis of Rat Mesenchymal Stem Cells through the Melanocortin-2 Receptor

Adrenocorticotropin Hormone (ACTH) is an endocrine hormone that is secreted by the pituitary and stimulates the secretion of cortisol from the adrenal cortex. It is among the several melanocortin peptide hormones that are derived from proopiomelanocortin (POMC) such as α-melanocyte stimulating hormone (α-MSH), γ-MSH and the endorphins. ACTH is also produced by cells outside the central nervous system and has been found to play a role in osteogenesis. Using mesenchymal stem cells (MSC) obtained from bone marrow of the Wystar Kyoto (WKY) rat, we confirmed that ACTH increases osteogenesis in a dosedependent manner. Immunoblot of crude membrane fractions was used to determine that rat MSC express three melanocortin receptors (MC-R); the MC2-R, MC3-R and MC5-R. To determine which of these receptors mediate ACTH-induced osteogenesis we used MC-R specific peptides and antagonists. Neither α-MSH, a strong agonist of the MC5-R nor γ2-MSH, a strong agonist of the MC3-R, increases osteogenesis in rat MSC. Additionally the MC3-R specific antagonist did not suppress ACTH-induced increases in osteogenesis. In addition, calcium flux was examined as a mechanism for ACTH action at the MC2-R. Consistent with MC2-R expression patterns in the MSC cultures, ACTH-induced transient increases in intracellular calcium were increased with dexamethasone treatment. Therefore the osteogenic effects of ACTH in rat MSC cultures are consistent with an MC2-R signaling mechanism. This pathway represents a new therapeutic target in the prevention and treatment of bone loss

Does Stress Make You Fat?

ACTH is a major hormone of the stress axis or hypothalamic-pituitary-adrenal (HPA) axis. It is derived from pro-opiomelanocortin (POMC) the precursor to the melanocortin family of peptides. POMC produces the biologically active melanocortin peptides via a series of enzymatic steps in a tissue-specific manner, yielding the melanocyte-stimulating hormones (MSHs), corticotrophin (ACTH) and β-endorphin. The melanocortin system plays an imperative role in energy expenditure, insulin release and insulin sensitivity. Bone marrow derived mesenchymal stem cells circulate in the blood stream and as progenitor cells have the potential to differentiate into many cell types such as osteoblasts, chondrocytes and adipocytes. Here we examine the effects of ACTH on the mouse D1 bonemarrow derived MSC. ACTH significantly increased lipid accumulation during the adipogenic differentiation of D1 cells in a concentration- dependent manner. ACTH also shifts the temporal pattern of D1 adipogenic differentiation to the left i.e. differentiation occurs earlier with ACTH treatment. No significant differences in protein expression of peroxisome proliferator-activated receptor gamma (PPAR-γ2), a regulating transcription factor of adipogenesis were found. Therefore the effects of ACTH are suggested to be mediated by an alternative pathway. Overall the results indicate a connection between increased adipose deposition and the elevated circulating ACTH associated with stress

CD105 Deficieny in Mouse Aorta-derived Mesenchymal Stem Cells Promotes An Enhanced Inflammatory Response to Lipolysaccharide.

Mesenchymal stem cells (MSCs) are being widely studied for their ability to regulate macrophage cell responses. Previous works have demonstrated that mouse aorta-derived MSC (mAo-MSC) support the macrophage inflammatory response. mAo-MSC have been characterized phenotypically for MSC-associated surface antigens and express CD90 and CD105 but do not express CD73. CD105, also known as endoglin, is a coreceptor in the TGFβ superfamily of receptors. Mouse adipose-derived MSC lacking CD105 have an increased capacity to regulate T-cells by reducing their proliferation while elevated CD105 expression is consistently associated with inflammatory disease. Therefore, we hypothesized that suppression of CD105 in mAo-MSC will reduce the immunosupportive capacity of the mAo-MSC. We used siRNA to reduce expression of CD105 in mAo- MSC and subsequently examined the effect of this deficiency on their response to lipopolysaccharide (LPS) and their ability to support the macrophage inflammatory response. Contrary to our hypothesis, CD105 deficient mAo-MSC cultured alone and in co-culture with macrophage secreted increased levels of the inflammatory indicators nitric oxide (NO) and interleukin 6 (IL-6) after exposure to LPS. The increase in NO and IL-6 observed in the co-cultures is additive and therefore points to the mAo- MSC as the primary origin. Overall our data suggest that CD105 acts as a regulator of the TLR-4 pathway and may represent an important target for modification of MSC to be used in therapeutics

Effect of circular motion exercise on bone modeling and bone mass in young rats: An animal model of isometric exercise

The aims of the study are to develop a non-invasive animal model of circular motion exercise and to evaluate the effect of this type of exercise on bone turnover in young rats. The circular motion exercise simulates isometric exercise using an orbital shaker that oscillates at a frequency of 50 Hz and is capable of speeds from 0-400 rpm. A cage is fixed on top of the shaker and the animals are placed inside. When the shaker is turned on, the oscillatory movement should encourage the animals to hold on to the cage and use various muscle forces to stabilize themselves. Rats at 8 weeks of age were trained on the shaker for 6 weeks and static and dynamic histomorphometric analyses were performed for the proximal tibial metaphysis and the tibial shaft. The exercise resulted in no significant effect on animal body weight, gastrocnemius muscle weight and femoral weight. Although the bone formation rate of cancellous and cortical periosteum was increased by the exercise, trabecular bone volume was decreased. The exercise increased periosteal and marrow perimeters and the cross-sectional diameter of cortical bone from medial to lateral without a significant increase in the cortical bone area. These results suggest that circular motion exercise under force without movement or additional weight loading will cause bone-modeling drift with an increase in bone turnover to reconstruct bone shape in adaptation to the demand in strength. Since there is no additional weight loading during circular motion exercise, the net mass of bone is not increased. The bone mass lost in trabecular bone could possibly be due to a re-distribution of mineral to the cortical bone

The Stress of Public Speaking Increases Cortisol Levels in Undergraduates: Is increased Preparation Really the Best Remedy?

Perceived stress is prevalent among the undergraduate population. When this stress persists, it has the potential to lead to mental health illnesses. Recent research shows 85% of students experience overwhelming anxiety from academic pressures. Physiologically, during stressful events, cortisol levels rise in the body which disrupts homeostasis. The anticipation prior to a class presentation, a form of public speaking, is a common source of perceived stress among undergraduates. The focus of this experiment was to determine if there is a correlation between factors such as increased preparedness, sleep, level of understanding, perceived anxiety and physiological stress parameters. Twenty-eight student volunteers with an impending oral presentation were enrolled from both 100- level and 200-level undergraduate courses. At baseline and on the day of the presentation, salivary cortisol, heart rate, and blood pressure were measured. The participants were also asked to complete the Beck’s Anxiety Inventory (BAI). Compared to baseline, cortisol levels on the day of the presentation were significantly increased in both groups. The change in salivary cortisol levels did not correlate with the number of hours spent preparing, the level of understanding nor hours of sleep the night before the presentation. However, the analyses revealed a trend toward an inverse correlation between the self-reported level of understanding and change in cortisol levels. Essentially, students who felt ambiguous toward their level of understanding of their presentation experienced lower changes in cortisol levels when compared to those students who reported a stronger understanding of the material. This study confirms that undergraduates’ perceived stress in anticipation of public speaking does manifest in significantly elevated cortisol levels. It does not provide a link between increased preparation and reduction of stress parameters. Future studies could focus on alternative methods such as mindfulness and meditation and their efficacy in reducing undergraduate stress associated with public speaking

Vascular Tissue-resident Mesenchymal Stem Cells; Friend or Foe?

Mesenchymal stem cells (MSC) are ideal candidates for stem cell-based therapies of vascular inflammation. They are progenitor cells that can replace damaged cells and they can modulate immune response cells. MSC from bone marrow and adipose tissue regulate inflammation by promoting the switch of macrophage cell from an inflammatory to an anti-inflammatory phenotype. Much less is known about the tissue resident MSC’s and their interaction with macrophage cells. We hypothesized that aortaderived mesenchymal stem cells (mAo MSC) would also promote the expression of the antiinflammatory phenotype among macrophage cells. The interaction of mAo MSC with the macrophage was examined by co-culturing the cells and exposing them to the inflammatory mediator, lipopolysaccharide (LPS). A bone marrow derived MSC cell line was used as a control. Nitric oxide (NO) and the tumor necrosis factor-α (TNFα) and interleukin-12 (IL-12) cytokines were measured using Griess reaction and ELISA assay respectively. The impact of the interaction on phagocytosis was measured using zymosan-A. We found that bone marrow derived MSC, when in co-culture with macrophage cells, performed as expected and suppressed NO, TNFα, and IL-12 production. Unexpectedly, the mAo MSC enhanced NO and TNFα production by the macrophage cells, which is indicative of the inflammatory phenotype; IL-12 production by macrophage remained unchanged. Macrophage cell phagocytic activity was significantly increased by contact with mAo MSC, which represents enhancement of the anti-inflammatory phenotype