In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

Porto, Isabel Maria; Rocha, Lenaldo Branco; Rossi, Marcos A.; Gerlach, Raquel Fernanda

doi:10.1369/jhc.2009.952127

Cited by 16 publications

(23 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Detection of gelatinolytic activity in the tissue sections was adapted from [30]. Briefly, DQ™ Gelatin from pig skin (1 mg/ml) (Molecular Probes), a fluorescein-conjugated gelatin in which fluorescence is quenched, was diluted (1/50) in reaction buffer (50 mM Tris-HCl pH 7,6, 150 mM NaCl, 5 mM CaCl 2 ) and poured onto the surface of the tissue sections, covered with Parafilm® to avoid evaporation, and incubated in the dark for 4 h at 37°C in a humidified chamber.…”

Section: Methodsmentioning

confidence: 99%

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

et al. 2013

View full text Add to dashboard Cite

The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

show abstract

Section: Methodsmentioning

confidence: 99%

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

et al. 2013

View full text Add to dashboard Cite

show abstract

“…This method is more sensitive than other methods, and due to uncompromised histology provides more detailed information on the localization of gelatinolytic activity. Since its introduction, it has been applied repeatedly with encouraging results (Frederiks and Mook 2004;Porto et al 2009;Wang and Tsirka 2005;Yan and Blomme 2003).…”

Section: Introductionmentioning

confidence: 99%

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Gkantidis

Katsaros

Chiquet

2012

Histochem Cell Biol

View full text Add to dashboard Cite

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple doublelabeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.Keywords Mouse embryo Á Craniofacial development Á Matrix metalloproteinase Á Basement membrane Á In situ zymography Á Dye-quenched gelatin Á Immunofluorescence

show abstract

Section: Resultsmentioning

confidence: 99%

“…ROIs were drawn around plaques and quantified for relative fluorescence to determine plaque targeting using ImageJ. Collagenase activity in the atherosclerotic lesions was determined using the DQ Collagenase Assay Kit (ThermoFisher, Waltham, MA, USA) following the manufacturer's protocol and previous methods . MFI for individual lesions was measured with ImageJ to analyze collagenase.…”

Section: Methodsmentioning

confidence: 99%

“…In order to verify the fibrous cap thickening by MCG PAMs was due to collagenase inhibition, MMP collagenase activity within the aortic atherosclerotic lesions was measured via in situ zymography. [58][59][60][61] Unfixed cryostat sections of aortic arches with atherosclerotic lesions were incubated with DQ-gelatin, which fluoresces when cleaved by collagenase. In correlation with cap thickness measurements, lesions in mice treated with MCG PAMs exhibited the least amount of fluorescence and collagenase activity per lesion (20.9 ± 7.3 mean fluorescence intensity (MFI)/lesion), followed by SCG PAM-and PBS-treated mice (47.0 ± 21.0 MFI/lesion and 55.5 ± 16.1 MFI/lesion, respectively).…”

Section: Mcg Pams Increase Fibrous Cap Thickness For Atherosclerosis mentioning

confidence: 99%

See 1 more Smart Citation

Collagenase‐Cleavable Peptide Amphiphile Micelles as a Novel Theranostic Strategy in Atherosclerosis

Chin

Poon

Trac

et al. 2020

Advanced Therapeutics

View full text Add to dashboard Cite

Atherosclerosis is an inflammatory disease characterized by plaques that can cause sudden myocardial infarction upon rupture. Such rupture‐prone plaques have thin fibrous caps due to collagenase degradation, and a noninvasive diagnostic tool and targeted therapy that can identify and treat vulnerable plaques and may inhibit the onset of acute cardiac events. Toward this goal, monocyte‐binding, collagenase‐inhibiting, and gadolinium‐modified peptide amphiphile micelles (MCG PAMs) are developed. Monocyte chemoattractant protein‐1 (MCP‐1) binds to C‐C chemokine receptor‐2 expressed on pathological cell types present within plaques. Through the peptide binding motif of MCP‐1, MCG PAMs bind to monocytes and vascular smooth muscle cells in vitro. Moreover, using magnetic resonance imaging, MCG PAMs show enhanced targeting and successful detection of plaques in diseased mice in vivo and act as contrast agents for molecular imaging. Through the collagenase‐cleaving peptide sequence of collagen [VPMS‐MRGG], MCG PAMs can compete for collagenases that degrade the fibrous cap of plaques, providing therapy. MCG PAM‐treated mice show increased fibrous cap thickness by 61% and 113% histologically compared to nontargeting micelle‐ or PBS‐treated mice (p = 0.0075 and 0.001, respectively). Overall, this novel multimodal nanoparticle offers new theranostic opportunities for noninvasive diagnosis and treatment of atherosclerotic plaques.

show abstract

In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

Cited by 16 publications

References 56 publications

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Collagenase‐Cleavable Peptide Amphiphile Micelles as a Novel Theranostic Strategy in Atherosclerosis

Contact Info

Product

Resources

About