Synthesis of a pH-Sensitive Nitrilotriacetic Linker to Peptide Transduction Domains To Enable Intracellular Delivery of Histidine Imidazole Ring-Containing Macromolecules

Intracellular delivery of functional macromolecules using peptide transduction domains (PTDs) is an exciting technology with both experimental and therapeutic applications. Recent data indicate that PTD-mediated transduction occurs via fluid-phase macropinocytosis involving an intracellular pH drop to ∼5. Nitrilotriacetic acid (NTA)-coordinated metals avidly bind hexahistidine-tagged macromolecules, including peptides and proteins. Histidine’s imidazole ring has a p<i>K</i>_a of 6, making this an attractive target for the biological pH drop of PTD-mediated macropinocytotic delivery. The objective of this study was to develop a pH-sensitive PTD delivery peptide (NTA₃-PTD). We demonstrate the <i>in vitro</i> function of this novel peptide by delivering fluorescently labeled peptides (1.6 kDa) and functional enzymes, β-galactosidase (119 kDa) and Cre recombinase (37 kDa). Furthermore, the NTA₃-PTD peptide was able to deliver functional Cre recombinase in an <i>in vivo</i> mouse model

Obesity is associated with increased levels of circulating MPs and increased adipose activity of Rho associated kinase and caspase 3

<p>Annexin V positive MPs from (A) ob control or ob/ob mice. Blood was collected by cardiac puncture and PFP was obtained as detailed in methods section. Annexin V positive MPs were analyzed by flow cytometry (n = 5 each group). (B) Dynamic light scattering analysis and Transmission Electron Microscopy of isolated circulating MPs. (C) Western blot analysis of perilipin A levels in MPs isolated from mouse PFP in WT mice on a regular chow diet and ob/ob mice (n = 3 each group). Perilipin A abundance in MPs isolated from mouse PFP correlated to: (D) total mouse body weights and (E) weight of extracted mouse epididymal fat pads. (F) Western blots of p-MYPT1, total MYPT1/2, cleaved (active) caspase 3, total caspase 3, and actin levels in subcutaneous and epididymal adipose tissue lysates from ob control or ob/ob mice. Quantification of Western blots of (G) cleaved caspase 3 and (H) p-MYPT in ob control and ob/ob mice adipose tissues normalized to actin levels. Values represent mean ± S.E.M. *P < 0.05; ***P < 0.001 compared to respective controls.</p

Microparticles Release by Adipocytes Act as “Find-Me” Signals to Promote Macrophage Migration

<div><p>Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical “find-me” signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel “find me” signals that contributes to macrophage infiltration associated with obesity.</p></div

Adipocyte-derived MPs mediates attraction of macrophages in vitro and in vivo.

<p>(A) In vitro chemotaxis assay of MPs-free supernatant and MPs from adipocytes treated with palmitic acid. Values represent mean ± S.D. ***P < 0.001 compared to controls. (B-F) C57BL/6 mice were injected intraperitoneally with 1x10⁶ adipocyte-derived MPs (isolated from 3T3-L1 adipocytes treated with 0.5 mM palmitic acid or without palmitic acid) or controls (n = 3 each group). (B) Four days post injection infiltrated cells were isolated from peritoneal cavity by lavage and counted. (C-F) flow cytometry analysis of infiltrated cells. The infiltrated cells were stained by CD45 (leukocyte common antigen) (C), CD11b (monocytes) (D), F4/80 (macrophages) (E), or Ly6G (neutrophils) (F). Values represent mean ± S.E.M. (D) In vivo macrophages migration. C57BL/6 mice were injected intraperitoneally with 1x10⁶ palmitic acid-derived MPs or vehicle alone (n = 5 per group). Three days post injection; macrophages were isolated from peritoneal cavity by lavage. Number of macrophages present in peritoneal cavity was counted. Values represent mean ± S.E.M.</p

Transplanted circulating MPs from ob/ob mouse lead to monocyte activation in the blood and macrophage infiltration in the adipose tissue.

<p>(A) Scheme of transplanted experiment; ob/ob platelet free plasma was collected, circulating MPs were purified and injected into the WT mouse. (B) Dot plot analysis of the entire leukocyte population in blood resulting from the mock, ob ctrl (control) MP, and ob/ob MP injections, respectively. X-axis indicates CD11b intensity and Y-axis indicates Ly6C intensity. (C-D) Flow cytometry analysis of monocyte (CD11b⁺-Ly6C^high) percentage (C) and activated monocyte (CD11b⁺-Ly6C^high-CD204⁺) percentage (D) in blood resulting from the mock, ob ctrl MP, or ob/ob MP injections (n = 4 each group). (E) Flow cytometry analysis of infiltrated monocytes (CD11b⁺-Ly6C^high) percentage in epididymal (Epi) adipose tissue from the mock, ob ctrl MP, or ob/ob MP injections. P<0.4. Values represent mean ± S.E.M. *P < 0.05; **P < 0.01 compared to ob ctrl MPs as a control.</p

Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease

<div><p>Background & Aim</p><p>Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.</p><p>Design</p><p>Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.</p><p>Results</p><p>We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r² = 0.64, p<0.05), fibrosis (r² = 0.66, p<0.05) and pathological angiogenesis (r² = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192 - two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.</p><p>Conclusions</p><p>These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.</p></div

Attraction of monocytes and macrophages to supernatants of mature adipocytes exposed to palmitic acid.

<p>Migration of (A) RAW264.7 cells or (B) primary mouse macrophages through a transwell (8 μm pore size) to supernatants from untreated (control) or treated differentiated mature adipocytes with 0.5mM palmitic acid. MCP-1 (50 ng/ml) was used as positive control. (C) Adipocytes were treated with 0.5 mM palmitic acid in the absence or presence of 0.025 U/ml apyrase, 0.5 U/ml phospholipase-D or 50 μg/ml control IgG or MCP-1 neutralizing antibody. Macrophages that migrated to the lower chamber were stained with DAPI and the number of cells was counted under fluorescence microscopy. Values represent as mean ± S.D. of representative experiment. * P < 0.05; ** P < 0.01; ***P < 0.001 compared to controls.</p

Caspase 3 activation is required for attraction of macrophages to stressed adipocytes.

<p>Caspase-3 activity assay in adipocytes treated with (A) a range of doses of palmitic acid (0.1 to 1 mM) in the absence or presence of a selective caspase 3 inhibitor (Ac-DEVD-CHO). Differentiated adipocytes were plated on black 96-well plate for 2 hrs followed by the different treatments for additional 4 hrs. Caspase-3 activity assay was determined by the Apo-One Homogeneous Caspase 3/7 fluorescent assay as described under Experimental Procedures. Assays were performed on three replicates for each treatment. Values represent as mean ± S.D. ** P < 0.01; ***P < 0.001 compared to controls. Transmigration of primary mouse macrophages to supernatants from differentiated adipocytes treated with (C) palmitic acid, in the absence or presence of increasing doses of the caspase-3 inhibitor was assessed. Values represent as mean ± S.D. ***P < 0.001 compared to stressor alone (no caspase inhibitor).</p

MPs are released by stressed adipocytes in a caspase 3 and Rho-associated kinase dependent manner.

<p>(A-D) Characterization of adipocyte-derived MPs. (A) Morphology of the differentiated 3T3-L1 adipocytes treated with control, 0.5 mM palmitic acid. (B) The number of Annexin V positive MPs was quantitated by flow cytometry. (C) Dynamic light scattering analysis and Transmission Electron Microscopy of isolated MPs. Isolated MPs were measured by Zetasizer and analyzed using intensity. (D) Western Blot analysis of MPs released by adipocytes. Isolated MPs were fractionated by SDS-PAGE and probed with FABP4, MCP-1, Chemerin, Adiponectin and Perilipin antibody. (E-F) Differentiated adipocytes were incubated with or without palmitic acid in the absence or presence of a selective caspase-3 inhibitor (E), or a range of doses of two different Rho associated kinase inhibitors (Y27632 and fasudil) (F) for up to 12 hrs. Supernatants were then collected and MPs isolated by ultracentrifugation as detailed in methods section. (G) Morphology of isolated mouse primary adipocytes. (H) Number of annexin V positive mouse primary adipocyte-derived MPs assessed by flow cytometry. Values represent mean ± S.D. * P < 0.5; ** P < 0.01; ***P < 0.001 compared to controls.</p

Protein identified by LC-MS/MS in circulating extracellular vesicles isolated from CDAA-induced NASH mice.

<p><b>a -</b> Significant MS/MS number of peptides identified in blood EVs from CDAA-fed mice.</p><p><b>b -</b> Significant MS/MS absolute % of coverage calculated in blood EV from CDAA-fed mice.</p><p><b>c -</b> Significant MS/MS spectral counts identified in blood EVs from CDAA-fed mice.</p><p>Protein identified by LC-MS/MS in circulating extracellular vesicles isolated from CDAA-induced NASH mice.</p