Fluorescence-Based Laser Capture Microscopy Technology Facilitates Identification of Critical In Vivo Cytomegalovirus Transcriptional Programs

The endometrium is a dynamic tissue, demonstrating cyclical growth/remodelling in preparation for implantation. In mice, seminal constituents trigger mechanisms to prepare the endometrium, a process dubbed ‘seminal priming’ that modifies immune system components and mediates endometrial remodelling in preparation for pregnancy. An array of cytokines has been reported to mediate this interaction, although much of the literature relates toin vitrostudies on isolated endometrial epithelial cells. This study measured changes in immune-related gene expression in endometrial epithelial and stromal cellsin vivofollowing natural mating. CD1 mice were naturally mated and sacrificed over the first 4 dayspost-coitum(n=3 each day). Endometrial epithelial and stromal compartments were isolated by laser capture microdissection. Labelled cRNA was generated and hybridised to genome-wide expression microarrays. Pathway analysis identified several immune-related pathways active within epithelial and stromal compartments, in particular relating to cytokine networks, matrix metalloproteinases and prostaglandin synthesis. Cluster analysis demonstrated that the expression of factors involved in immunomodulation/endometrial remodelling differed between the epithelial and stromal compartments in a temporal fashion. This study is the first to examine the disparate responses of the endometrial epithelial and stromal compartments to seminal plasmain vivoin mice, and demonstrates the complexity of the interactions between these two compartments needed to create a permissive environment for implantation.

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Effect of mechanical stretch on HIF-1α and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00284.2005 ◽

2005 ◽

Vol 289 (3) ◽

pp. H1315-H1320 ◽

Cited By ~ 46

Author(s):

Malgorzata Milkiewicz ◽

Tara L. Haas

Keyword(s):

Endothelial Cells ◽

Laser Capture Microdissection ◽

Skeletal Muscles ◽

Hypoxia Inducible Factor ◽

Fold Increase ◽

Mechanical Stretch ◽

Specific Gene ◽

Capillary Endothelial Cells ◽

Laser Capture

Under physiological nonhypoxic conditions, angiogenesis can be driven by mechanical forces. However, because of the limitations of the specific gene expression analysis of microvessels from in vivo experiments, the mechanisms regulating the coordinated expression of angiogenic factors implicated in the process remain intangible. In this study, the technique of laser capture microdissection (LCM) was adapted for the study of angiogenesis in skeletal muscles. With a combination of LCM and real-time quantitative PCR it was demonstrated that capillary endothelial cells produce matrix metalloproteinase (MMP)-2 and that mechanical stretch of capillaries within muscle tissue markedly increases MMP-2 mRNA (2.5-fold increase vs. control; P < 0.05). In addition, we showed that transcription factor hypoxia-inducible factor (HIF)-1α expression was 13.5-fold higher in capillaries subjected to stretch compared with controls ( P < 0.05). These findings demonstrate the feasibility of this approach to study angiogenic gene regulation and provide novel evidence of HIF-1α induction in stretched capillary endothelial cells.

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Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

Molecular Biology of the Cell ◽

10.1091/mbc.e06-12-1069 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2864-2872 ◽

Cited By ~ 13

Author(s):

Kelly J. Perkins ◽

Utpal Basu ◽

Murat T. Budak ◽

Caroline Ketterer ◽

Santhosh M. Baby ◽

...

Keyword(s):

Promoter Activity ◽

Molecular Mechanisms ◽

Neuromuscular Junctions ◽

Protein Product ◽

Mrna Levels ◽

Laser Capture Microscopy ◽

Erk Phosphorylation ◽

Duchenne's Muscular Dystrophy

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPα/β to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest “repressing repressors” as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

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Cambridge Healthtech Institute’s 3rd Annual Laser Capture Microscopy: Cutting Edge Applications

Expert Review of Molecular Diagnostics ◽

10.1586/14737159.5.4.487 ◽

2005 ◽

Vol 5 (4) ◽

pp. 487-491 ◽

Cited By ~ 1

Author(s):

Vasker Bhattacherjee ◽

Robert Greene ◽

M Michele Pisano

Keyword(s):

Cutting Edge ◽

Laser Capture Microscopy ◽

Laser Capture

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Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma

Oncogene ◽

10.1038/sj.onc.1206614 ◽

2003 ◽

Vol 22 (25) ◽

pp. 3964-3976 ◽

Cited By ~ 56

Author(s):

Tanja Xenia Pedersen ◽

Chidchanop Leethanakul ◽

Vyomesh Patel ◽

David Mitola ◽

Leif Røge Lund ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Laser Capture Microdissection ◽

Genomic Profiling ◽

Similarities And Differences ◽

Laser Capture

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Laser capture microscopy as a tool for the assessment of lineage-specific chimaerism from archived blood and bone marrow films

British Journal of Haematology ◽

10.1111/j.1365-2141.2006.06470.x ◽

2007 ◽

Vol 136 (4) ◽

pp. 677-678 ◽

Cited By ~ 1

Author(s):

Julie Richards ◽

G. Yvonne Morgan ◽

Laurence Pearce ◽

Piers E. M. Patten ◽

Ghulam J. Mufti ◽

...

Keyword(s):

Bone Marrow ◽

Laser Capture Microscopy ◽

Laser Capture

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Human Neutrophil Peptides 1-3 – Early Markers in Development of Colorectal Adenomas and Carcinomas

Disease Markers ◽

10.1155/2008/693937 ◽

2008 ◽

Vol 25 (2) ◽

pp. 123-129 ◽

Cited By ~ 10

Author(s):

Henning Mothes ◽

Christian Melle ◽

Günther Ernst ◽

Roland Kaufmann ◽

Ferdinand von Eggeling ◽

...

Keyword(s):

Human Neutrophil ◽

Tumour Growth ◽

Tumour Response ◽

Colorectal Adenomas ◽

Cancer Stage ◽

Tumour Stage ◽

Laser Capture Microscopy ◽

Early Markers ◽

Laser Capture

Expression of Human Neutrophil Peptides (HNP) 1–3 was recently found to be associated with development of colorectal cancer. Raised defensin-expression in tumours is believed to stem from increased infiltration of neutrophils into tumour environment.To further specify the role of α-defensins in tumourigenesis and progression, HNP1–3 were analyzed in colorectal adenomas and carcinomas of 87 patients and quantified in relation to cancer stage and grading. Using the ProteinChip arrays, HNP1–3 were found upregulated in both colorectal adenomas and carcinomas. By combining the array with Laser capture microscopy we were able to confirm that HNP1–3 are expressed by tumour cells but not by neutrophils or other tumour invading cells. These findings suggest that α-defensins are more likely to contribute to tumour growth than they are to mount an effective host anti-tumour response. However, the amount of HNP-expression was not found to be related to tumour stage, grading, and serological tumour markers.

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