Point of care assessment of melanoma tumor signaling and metastatic burden from µNMR analysis of tumor fine needle aspirates and peripheral blood.

This study evaluates µNMR technology for molecular profiling of tumor fine needle aspirates and peripheral blood of melanoma patients. In vitro assessment of melanocyte (MART-1, HMB45) and MAP kinase signaling (pERK, pS6K) molecule expression was performed in human cell lines, while clinical validation was performed in an IRB-approved study of melanoma patients undergoing biopsy and blood sampling. Tumor FNA and blood specimens were compared with BRAF genetic analysis and cross-sectional imaging. µNMR in vitro analysis showed increased expression of melanocyte markers in melanoma cells as well as increased expression of phosphorylated MAP kinase targets in BRAF-mutant melanoma cells. Melanoma patient FNA samples showed increased pERK and pS6K levels in BRAF mutant compared with BRAF WT melanomas, with µNMR blood circulating tumor cell level increased with higher metastatic burden visible on imaging. These results indicate that µNMR technology provides minimally invasive point-of-care evaluation of tumor signaling and metastatic burden in melanoma patients.

Myeloperoxidase Inhibition Improves Ventricular Function and Remodeling After Experimental Myocardial Infarction.

PF-1355 is an oral myeloperoxidase (MPO) inhibitor that successfully decreased elevated MPO activity in mouse myocardial infarction models. Short duration PF-1355 treatment for 7 days decreased the number of inflammatory cells and attenuated left ventricular dilation. Cardiac function and remodeling improved when treatment was increased to 21 days. Better therapeutic effect was further achieved with early compared with delayed treatment initiation (1 h vs. 24 h after infarction). In conclusion, PF-1355 treatment protected a mouse heart from acute and chronic effects of MI, and this study paves the way for future translational studies investigating this class of drugs in cardiovascular diseases.

Targeting Interleukin-1ß Reduces Leukocyte Production After Acute Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) is an ischemic wound that recruits millions of leukocytes. MI-associated blood leukocytosis correlates inversely with patient survival, yet the signals driving heightened leukocyte production after MI remain incompletely understood. METHODS AND RESULTS: With the use of parabiosis surgery, this study shows that soluble danger signals, among them interleukin-1ß, increase bone marrow hematopoietic stem cell proliferation after MI. Data obtained in bone marrow reconstitution experiments reveal that interleukin-1ß enhances hematopoietic stem cell proliferation by both direct actions on hematopoietic cells and through modulation of the bone marrow's hematopoietic microenvironment. An antibody that neutralizes interleukin-1ß suppresses these effects. Anti-interleukin-1ß treatment dampens the post-MI increase in hematopoietic stem cell proliferation. Consequently, decreased leukocyte numbers in the blood and infarct reduce inflammation and diminish post-MI heart failure in ApoE(-/-) mice with atherosclerosis. CONCLUSIONS: The presented insight into post-MI bone marrow activation identifies a mechanistic target for muting inflammation in the ischemically damaged heart.

Myocardial Infarction Activates CCR2(+) Hematopoietic Stem and Progenitor Cells.

Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.

Automated motion artifact removal for intravital microscopy, without a priori information.

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

Magnetic ligation method for quantitative detection of microRNAs.

A magnetic ligation method is utilized for the detection of microRNAs among a complex biological background without polymerase chain reaction or nucleotide modification. The sandwich probes assay can be adapted to analyze a panel of microRNAs associated with cardiovascular diseases in heart tissue samples.

In vivo silencing of the transcription factor IRF5 reprograms the macrophage phenotype and improves infarct healing.

OBJECTIVES: The aim of this study was to test whether silencing of the transcription factor interferon regulatory factor 5 (IRF5) in cardiac macrophages improves infarct healing and attenuates post-myocardial infarction (MI) remodeling. BACKGROUND: In healing wounds, the M1 toward M2 macrophage phenotype transition supports resolution of inflammation and tissue repair. Persistence of inflammatory M1 macrophages may derail healing and compromise organ functions. The transcription factor IRF5 up-regulates genes associated with M1 macrophages. METHODS: Here we used nanoparticle-delivered small interfering ribonucleic acid (siRNA) to silence IRF5 in macrophages residing in MIs and in surgically-induced skin wounds in mice. RESULTS: Infarct macrophages expressed high levels of IRF5 during the early inflammatory wound-healing stages (day 4 after coronary ligation), whereas expression of the transcription factor decreased during the resolution of inflammation (day 8). Following in vitro screening, we identified an siRNA sequence that, when delivered by nanoparticles to wound macrophages, efficiently suppressed expression of IRF5 in vivo. Reduction of IRF5 expression, a factor that regulates macrophage polarization, reduced expression of inflammatory M1 macrophage markers, supported resolution of inflammation, accelerated cutaneous and infarct healing, and attenuated development of post-MI heart failure after coronary ligation as measured by protease targeted fluorescence molecular tomography-computed tomography imaging and cardiac magnetic resonance imaging (p < 0.05). CONCLUSIONS: This work identified a new therapeutic avenue to augment resolution of inflammation in healing infarcts by macrophage phenotype manipulation. This therapeutic concept may be used to attenuate post-MI remodeling and heart failure.