Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

Hadler‐Olsen, Elin; Kanapathippillai, Premasany; Berg, Eli; Svineng, Gunbjørg; Winberg, Jan‐Olof; Uhlin‐Hansen, Lars

doi:10.1369/jhc.2009.954354

Cited by 63 publications

(72 citation statements)

References 48 publications

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“…This suggests that vBM components could be locally degraded. Indeed, in situ zymography 21 on RipTag2 tumour slices revealed gelatinase activity in the regions that contained F-actin ring-like structures and were simultaneously devoid of laminin staining (Fig. 2b and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 97%

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis

et al. 2014

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The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6β1. In contrast, the binding of α6β1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6β1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.

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Section: Resultsmentioning

confidence: 97%

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis

et al. 2014

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“…Collagenase activity was measured using in situ zymography of zinc-buffered, formalin-fixed, paraffin-embedded liver tissue as described elsewhere (Hadler-Olsen et al , 2010; Kumar et al , 2014). Zinc-buffered formalin fixative has been shown to preserve the morphological and functional properties of tissues, including the enzymatic activity of matrix metalloproteinases (Beckstead, 1994), which are zinc-dependent endopeptidases that cleave extracellular matrix proteins.…”

Section: Methodsmentioning

confidence: 99%

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice

Lamb

Cholico

et al. 2016

Toxicology and Applied Pharmacology

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimental liver fibrosis. To elicit fibrosis, C57BL6/ male mice were treated twice weekly for 8 weeks with 0.5 ml/kg carbon tetrachloride (CCl4). TCDD (20 μg/kg) or peanut oil (vehicle) was administered once a week during the last 2 weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl4-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homologue-1, TGF-β1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl4-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling.

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“…Four µm sections of ZBF-fixed and paraffin embedded tumours were analysed as previously described [70]. The contribution of enzymatic activity from gelatinolytic enzymes that were not metal dependent was assessed by incubating the sections in 20 mM of EDTA (a metalloproteinase inhibitor).…”

Section: Methodsmentioning

confidence: 99%

Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

et al. 2014

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BackgroundThe urokinase plasminogen activator receptor (uPAR) is associated with poor prognosis in oral squamous cell carcinoma (OSCC), and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells’ expression level of uPAR affected the activity of gelatinolytic enzymes.MethodsThe Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM) proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.ResultsWe found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.ConclusionsAlthough high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational modification of uPAR.

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Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

Cited by 63 publications

References 48 publications

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice

Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

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