Rat Mammary Extracellular Matrix Composition and Response to Ibuprofen Treatment During Postpartum Involution by Differential GeLC–MS/MS Analysis

O’Brien, Jenean; Vanderlinden, Lauren A.; Schedin, Pepper; Hansen, Kirk C.

doi:10.1021/pr3003744

Cited by 33 publications

(36 citation statements)

References 70 publications

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“…These findings suggest that intact activation of inflammatory pathways is important for the physiological rise in the peritendinous collagen synthesis that is normally seen with mechanical loading of tendon tissue. In rat tendon cells, anti-inflammatory medication did not affect expression of collagen, but did lead to upregulation of several collagenases (44), while in rat mammary, extracellular matrix anti-inflammatory medication reduced extracellular matrix protein content of tenascin C and laminin (32). It is important to note that, in the two latter studies, no systematic mechanical loading was applied, so although the results do provide some information regarding role of inflammatory pathways for matrix changes, it can only, to a limited degree, provide conclusive evidence in regards to the interaction between inflammation and mechanical loading.…”

Section: Role Of Inflammatory Mediators In Adaptation Of Healthy Tendmentioning

confidence: 87%

What is the impact of inflammation on the critical interplay between mechanical signaling and biochemical changes in tendon matrix?

Kjær

Bayer

Eliasson

et al. 2013

Journal of Applied Physiology

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Section: Role Of Inflammatory Mediators In Adaptation Of Healthy Tendmentioning

confidence: 87%

What is the impact of inflammation on the critical interplay between mechanical signaling and biochemical changes in tendon matrix?

Kjær

Bayer

Eliasson

et al. 2013

Journal of Applied Physiology

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“…Briefly, 30gs of sample was added to a solution of ammonium persulfate, bis-acrylamide, and TEMED to polymerize the samples in an Eppendorf tube. Samples were then reduced, alkylated, and digested with trypsin as described (12). Briefly, gel pieces were washed and then reduced with 5 mM DTT for 25 min at 64°C and alkylated with 20 mM Iodoacetamide in the dark at room temperature for 45 min.…”

Section: Methodsmentioning

confidence: 99%

“…Current relative quantification strategies (iTRAQ, Spectral Counting, dimethyl labeling, others) (11)(12)(13)(14)(15) perform well when the majority of protein in samples does not change, there are approximately equal increases and decreases in protein levels, or in cases where proteins that are known not to change in abundance can be used for normalization. However, normalization steps often employed have the potential to introduce experimental bias (16).…”

mentioning

confidence: 99%

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

Hill

Calle²,

Dzieciątkowska

et al. 2015

Molecular & Cellular Proteomics

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137

156

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The use of extracellular matrix (ECM) 1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotropesoluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. Molecular & Cellular Proteomics

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“…This hypothesis is supported by studies in which breast cancer cells transplanted into involuting mouse mammary fat pads grow and invade more rapidly than do cells transplanted into mammary glands of nulliparous mice (10)(11)(12). This postpartum transplantation model elegantly distinguished the microenvironmental influences of the involuting postpartum mammary gland versus the mammary gland at other reproductive stages (3,(10)(11)(12)(13)(14)(15). Thus, it is imperative to understand how the changing landscape of the postpartum breast accelerates cancer progression in order to design more effective therapies for patients suffering from ppBC or strategies to limit the alterations leading to ppBCs.…”

Section: Introductionmentioning

confidence: 90%

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution

Stanford¹,

Young²,

Hicks³

et al. 2014

J. Clin. Invest.

125

144

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qRT-PCR. Whole-tumor RNA was harvested with an RNeasy kit (QIAGEN), and cDNA was synthesized (High Capacity; Applied Biosystems) and amplified using the murine cDNA-specific primers (Integrated DNA Technologies) listed in Supplemental Methods, along with SYBR Green Supermix (Bio-Rad). The following primers were used: MRC1 (forward: 5′ CCCTCAGCAAGCGATGTGC 3′; reverse: 5′-GGATACTTGCCAGGT CCCCA-3′); iNOS (forward: 5′-GGAGCATCCCAAGTACGAGTGG-3′; reverse: 5′-CGGCC-CACTTCCTCCAG); IL10 (forward: 5′-GGCGCTGTCATC-GATTTCTCC; reverse: 5′-GGCCTTGTAGACACCTTGGTC); Tgfb1 (forward: 5′-CGCAACAACGCCATCTATGAG; reverse: 5′-CGG-GACAGCAATGGGGGTTC); IL4 (forward: 5′-GGTCACAGGAGAAGG-GACG; reverse: 5′-GCGAAGCACCTTGGAAGCC);, IL12b (forward: 5′-GGAGTGGGATGTGTCCTCAG; reverse: 5′-CGGGAGTCCAGTC-CACCTCT); CCL3 (forward: 5′-CCACTGCCCTTGCTGTTCTTCTCT; reverse: 5′-GGGTGTCAGCTCCATATGGCG); and Rplp0 (forward: 5′-TCCTATAAAAGGCACACGCGGGC; reverse: 5′-AGACGATGT-CACTCCAACGAGGACG). Target To generate apoptotic MCF7, cells were treated in suspension with 1 μm BKM120 plus 2 μm ABT-263 (both inhibitors from Selleck Chemicals) for 4 hours, washed 5 times with PBS to remove residual drug, and used directly for efferocytosis assays or for annexin V staining. For efferocytosis coculture assays, Raw264.7-GFP cells (10 4 /well) and PyVmT or MCF7 cells (72 hours after infection with Ad.mCherry and Ad.HS-V-TK) were seeded together in a monolayer in 24-well plates in 2% FBS and cultured for 24 hours prior to the addition PBS or gancyclovir. Cells were imaged at 8, 16, and 32 h after addition of gancyclovir. Cells were collected and counted under fluorescence after 32 hours of coculture. In some experiments, Raw264.7-GFP cells (10 4 / well) were seeded in a monolayer in 24-well plates and cultured for 24 hours prior to the addition of 10 3 live MCF7-mCherry or 10 3 dead MCF7-mCherry cells in serum-free media. Where indicated in the figures, BMS-777607 (1 μm) or a neutralizing goat anti-mouse MerTK antibody (AF591, 25 μg/ml; R&D Systems)(44) was added 2 hours prior to the addition of gancyclovir or 2 hours prior to the addition of dead MCF7 cells to macrophage monolayers. Live and dead MCF7 cells were similarly seeded without Raw264.7 cells as single cultures. Media were collected after 16 hours of coculture, passed through a 0.2-μm filter, and used neat (250 μl) to quantify murine IL-10 and IL-4 by ELISA (BioLegend) according to the manufacturer's protocol. Total remaining cells were collected after 16 hours of coculture, lysed, and RNA was collected using an RNeasy kit (QIAGEN). MethodsMice. All mice were inbred on an FVB background for more than 10 generations. WT FVB, MMTV PyVmT and MerTK -/-mice (67), originally referred to as Mer KD , were purchased from The Jackson Laboratory. Mice were genotyped by PCR of genomic DNA as previously described(30). Female virgin mice were randomized into 2 groups: (a) 1 group that remained virgin, and (b) 1 group that was bred from 42 to 44 days of age with WT male mice. Pregnancies were timed according to identification of a va...

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Rat Mammary Extracellular Matrix Composition and Response to Ibuprofen Treatment During Postpartum Involution by Differential GeLC–MS/MS Analysis

Cited by 33 publications

References 70 publications

What is the impact of inflammation on the critical interplay between mechanical signaling and biochemical changes in tendon matrix?

What is the impact of inflammation on the critical interplay between mechanical signaling and biochemical changes in tendon matrix?

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution

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