David Melton - Academia.edu (original) (raw)

Papers by David Melton

Physiological Genomics, 2016

Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different... more Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different stimulating conditions and environments. However, temporal patterns of miRNAs across multiple macrophage polarization phenotypes have not been defined. We determined miRNA expression in bone marrow-derived murine macrophages over multiple time points (0.5, 1, 3, 24 hours) following exposure to cytokines and/or LPS. We hypothesized that dynamic changes in miRNAs regulate macrophage phenotypes. Changes in macrophage polarization markers were detected as early as 0.5 and as late as 24 hours, however, robust responses for most markers occurred within 3 hours. In parallel, many polarization-specific miRNAs were also changed by 3 hours and expressed divergent patterns between M1 and M2a conditions, with increased expression in M1 (miR-155, 199a-3p, 214-3p, 455-3p, and 125a) or M2a (miR-511 and 449a). Specifically, miR-125a-5p exhibited divergent patterns; increased at 12 - 24 hours in M1 macrophages and decreasing trend in M2a. VEGF in the culture media of macrophages was dependent upon the polarization state, with greatly diminished VEGF in M2a compared to M1 macrophage culture media despite similar VEGF in cell lysates. Inhibition of miR-125a-5p in media only controls (MO) and M1 macrophages greatly increased expression and secretion of soluble VEGF receptor-1 (sVEGFR1) leading to diminished VEGF in the culture media; partially converting MO and M1 into an M2a phenotype. Thus, the divergent expression patterns of polarization-specific miRNAs led to the identification and demonstrated the regulation of a specific macrophage polarization phenotype, sVEGFR1 by inhibition of miR-125a-5p.

The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2015

We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; ho... more We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; however, female mice transiently increased adipocyte area within regenerated muscle in a sex hormone-dependent manner. We extended these observations to investigate the effect of aging and sex on sarcopenia and muscle regeneration. Cardiotoxin injury to the tibialis anterior muscle of young, middle, and old-aged C57Bl/6J male and female mice was used to measure regenerated myofiber cross-sectional area (CSA), adipocyte area, residual necrosis, and inflammatory cell recruitment. Baseline (uninjured) myofiber CSA was decreased in old mice of both sexes compared to young and middle-aged mice. Regenerated CSA was similar in male mice in all age groups until baseline CSA was attained but decreased in middle and old age female mice compared to young females. Furthermore, adipocyte area within regenerated muscle was transiently increased in young females compared to young males and these sex-dependent increases persisted in middle and old age female mice and were associated with increased Pparg. Young female mice had more pro-inflammatory monocytes/macrophages in regenerating muscle than young male mice and increased Sca-1(+)CD45(-)cells. In conclusion, sex and age influence pro-inflammatory cell recruitment, muscle regeneration, and adipocyte area following skeletal muscle injury.

Autoimmunity, Jan 31, 2015

Macrophages are important in vascular inflammation and environmental factors influence macrophage... more Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ + LPS (M1), IL-4 (M2a) or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5-3 h), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3-6 h) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fi...

Physiological Genomics, 2012

1 other HighWire hosted articles This article has been cited by [PDF] [Full Text] [Abstract] , Ma... more 1 other HighWire hosted articles This article has been cited by [PDF] [Full Text] [Abstract] , March 1, 2015; 47 (3): 45-57.

Journal of Biomedicine and Biotechnology, 2009

Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells withou... more Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of ∼4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at 15 • C, increased the coinfection rate to ∼10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to ∼25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or >50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.

The American Journal of Pathology, 2014

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11bdip... more Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11bdiphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b þ (CD90/B220/CD49b/NK1.1/Ly6G) À (F4/80/I-Ab/CD11c) À Ly6C þ/À ] accumulation occurred; interestingly, Ly6C þ and Ly6C À monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C þ macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b þ F4/80 þ cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b þ F4/80 þ Ly6C À CD301 À macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of antiinflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C À macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration. (Am J Pathol 2014, 184: 1167e1184; http://dx.

Physiological Genomics, 2016

Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different... more Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different stimulating conditions and environments. However, temporal patterns of miRNAs across multiple macrophage polarization phenotypes have not been defined. We determined miRNA expression in bone marrow-derived murine macrophages over multiple time points (0.5, 1, 3, 24 hours) following exposure to cytokines and/or LPS. We hypothesized that dynamic changes in miRNAs regulate macrophage phenotypes. Changes in macrophage polarization markers were detected as early as 0.5 and as late as 24 hours, however, robust responses for most markers occurred within 3 hours. In parallel, many polarization-specific miRNAs were also changed by 3 hours and expressed divergent patterns between M1 and M2a conditions, with increased expression in M1 (miR-155, 199a-3p, 214-3p, 455-3p, and 125a) or M2a (miR-511 and 449a). Specifically, miR-125a-5p exhibited divergent patterns; increased at 12 - 24 hours in M1 macrophages and decreasing trend in M2a. VEGF in the culture media of macrophages was dependent upon the polarization state, with greatly diminished VEGF in M2a compared to M1 macrophage culture media despite similar VEGF in cell lysates. Inhibition of miR-125a-5p in media only controls (MO) and M1 macrophages greatly increased expression and secretion of soluble VEGF receptor-1 (sVEGFR1) leading to diminished VEGF in the culture media; partially converting MO and M1 into an M2a phenotype. Thus, the divergent expression patterns of polarization-specific miRNAs led to the identification and demonstrated the regulation of a specific macrophage polarization phenotype, sVEGFR1 by inhibition of miR-125a-5p.

The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2015

We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; ho... more We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; however, female mice transiently increased adipocyte area within regenerated muscle in a sex hormone-dependent manner. We extended these observations to investigate the effect of aging and sex on sarcopenia and muscle regeneration. Cardiotoxin injury to the tibialis anterior muscle of young, middle, and old-aged C57Bl/6J male and female mice was used to measure regenerated myofiber cross-sectional area (CSA), adipocyte area, residual necrosis, and inflammatory cell recruitment. Baseline (uninjured) myofiber CSA was decreased in old mice of both sexes compared to young and middle-aged mice. Regenerated CSA was similar in male mice in all age groups until baseline CSA was attained but decreased in middle and old age female mice compared to young females. Furthermore, adipocyte area within regenerated muscle was transiently increased in young females compared to young males and these sex-dependent increases persisted in middle and old age female mice and were associated with increased Pparg. Young female mice had more pro-inflammatory monocytes/macrophages in regenerating muscle than young male mice and increased Sca-1(+)CD45(-)cells. In conclusion, sex and age influence pro-inflammatory cell recruitment, muscle regeneration, and adipocyte area following skeletal muscle injury.

Autoimmunity, Jan 31, 2015

Macrophages are important in vascular inflammation and environmental factors influence macrophage... more Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ + LPS (M1), IL-4 (M2a) or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5-3 h), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3-6 h) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fi...

Physiological Genomics, 2012

1 other HighWire hosted articles This article has been cited by [PDF] [Full Text] [Abstract] , Ma... more 1 other HighWire hosted articles This article has been cited by [PDF] [Full Text] [Abstract] , March 1, 2015; 47 (3): 45-57.

Journal of Biomedicine and Biotechnology, 2009

Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells withou... more Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of ∼4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at 15 • C, increased the coinfection rate to ∼10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to ∼25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or >50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.

The American Journal of Pathology, 2014

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11bdip... more Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11bdiphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b þ (CD90/B220/CD49b/NK1.1/Ly6G) À (F4/80/I-Ab/CD11c) À Ly6C þ/À ] accumulation occurred; interestingly, Ly6C þ and Ly6C À monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C þ macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b þ F4/80 þ cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b þ F4/80 þ Ly6C À CD301 À macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of antiinflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C À macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration. (Am J Pathol 2014, 184: 1167e1184; http://dx.