isolation-human-endothelial-cells-from-normal-colon-colorectal

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

investigating-intestinal-barrier-breakdown-in-living-organoids

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Friedrich-Alexander University of Erlangen-Nuremberg

Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen

The large GTPase human guanylate binding protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation and is induced by interferon (IFN)-α and IFN-γ in endothelial cells (ECs). The aim of this study was to investigate whether GBP-1 is a marker of skin lesions in patients with cutaneous lupus erythematosus (CLE). Western blotting revealed that GBP-1 was in vitro induced by IFN-α and -γ in primary keratinocytes obtained from healthy controls. Moreover, we found that this protein was expressed by keratinocytes and ECs in primary and ultraviolet (UV)-induced skin lesions from patients with various subtypes of CLE, when compared to non-lesional skin. No GBP-1 expression was noted in skin biopsy specimens 24 or 72 h after UV irradiation prior to lesion formation in patients with CLE or in healthy control specimens with or without UV irradiation. Initial findings suggest that GBP-1 is not expressed in other skin diseases with different inflammatory aetiology, such as atopic dermatitis. We conclude that GBP-1 expression is closely associated with skin lesions in patients with CLE, suggesting a contribution of GBP-1 in the pathogenesis of this disease.

Increased expression of guanylate binding protein-1 in lesional skin of patients with cutaneous lupus erythematosus.

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins.

Intracellular trafficking of guanylate-binding proteins is regulated by heterodimerization in a hierarchical manner.

Antiangiogenic drugs have been used successfully for the treatment of colorectal cancer (CRC) and several other tumor types. Until recently, viable tumor endothelial cells (TEC) and normal endothelial cells of uninvolved colon tissue (NEC) from the same patient have not been available to optimize treatment strategies in vitro. Here, we describe a protocol for the isolation of TEC and NEC. These cells were isolated at a very high purity via magnetic cell sorting of tissue samples obtained from surgical specimens of patients suffering from CRC. Isolated TEC and NEC expressed CD31, CD105, VE-cadherin, VCAM-1, ICAM-1, and E-selectin, formed capillaries in basal membrane extract, and were able to take up acetylated LDL. They were negative for podoplanin, CD45, CD68, and CK-20, indicating blood vessel endothelial lineage. Expression of vWF was more pronounced in NEC cultures, whereas vWF was absent or only slightly expressed in all TEC cultures in vitro. Lower intracellular concentrations of vWF were also detected in TEC as compared to NEC at the tissue level. The latter finding demonstrated that differential features of TEC and NEC in vivo are stably perpetuated in culture. The isolated endothelial cell cultures may provide a useful in vitro model system to elucidate epigenetic effects on angiogenesis in cancer and to optimize antiangiogenic therapy.

Isolation of endothelial cells from human tumors.

Proinflammatory cytokines induce guanylate-binding protein 1 (GBP-1) protein expression in intestinal epithelial tissues. GBP-1 has been described as influencing a number of cellular processes important for epithelial homeostasis, including cell proliferation. However, many questions remain as to the role of GBP-1 in intestinal mucosal homeostasis. We therefore sought to investigate the function of proinflammatory cytokine-induced GBP-1 during intestinal epithelial cell proliferation. Through the use of complementary GBP-1 overexpression and small interfering RNA-mediated knockdown studies, we now show that GBP-1 acts to inhibit pro-mitogenic β-catenin/T cell factor (TCF) signaling. Interestingly, proinflammatory cytokine-induced GBP-1 was found to be a potent suppressor of β-catenin protein levels and β-catenin serine 552 phosphorylation. Neither glycogen synthase kinase 3β nor proteasomal inhibition alleviated GBP-1-mediated suppression of cell proliferation or β-catenin/TCF signaling, indicating a non-canonical mechanism of β-catenin inhibition. Together, these data show that cytokine-induced GBP-1 retards cell proliferation by forming a negative feedback loop that suppresses β-catenin/TCF signaling.

IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of β-catenin/TCF signaling.

The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.

GBP-1 acts as a tumor suppressor in colorectal cancer cells.

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.

Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

Gout is characterized by an acute inflammatory reaction and the accumulation of neutrophils in response to monosodium urate (MSU) crystals. Inflammation resolves spontaneously within a few days, although MSU crystals can still be detected in the synovial fluid and affected tissues. Here we report that neutrophils recruited to sites of inflammation undergo oxidative burst and form neutrophil extracellular traps (NETs). Under high neutrophil densities, these NETs aggregate and degrade cytokines and chemokines via serine proteases. Tophi, the pathognomonic structures of chronic gout, share characteristics with aggregated NETs, and MSU crystals can induce NETosis and aggregation of NETs. In individuals with impaired NETosis, MSU crystals induce uncontrolled production of inflammatory mediators from neutrophils and persistent inflammation. Furthermore, in models of neutrophilic inflammation, NETosis-deficient mice develop exacerbated and chronic disease that can be reduced by adoptive transfer of aggregated NETs. These findings suggest that aggregated NETs promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation.

Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines.

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using (15) N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.

Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS.

Interferon (IFN)-γ is a central pathogenesis factor in inflammatory bowel disease (IBD) with pleiotropic effects on many different cell types. However, as yet, the immune modulatory functions of IFN-γ in IBD have been predominantly investigated. Based on previous studies showing that IFN-γ acts antiangiogenic in colorectal carcinoma, we investigated the effects of IFN-γ on the vascular system in IBD.

Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice.

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.

Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis.

Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton.

ArticleTotal : 2	Year
Publication title Cited by 9	2018
Publication title Cited by 13	2020

Article	Year
Increased expression of guanylate binding protein-1 in lesional skin of patients with cutaneous lupus erythematosus. Experimental dermatology\| PubMed ID: 21121962	2011
Intracellular trafficking of guanylate-binding proteins is regulated by heterodimerization in a hierarchical manner. PloS one\| PubMed ID: 21151871	2010
Isolation of endothelial cells from human tumors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21516410	2011
IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of β-catenin/TCF signaling. Mucosal immunology\| PubMed ID: 22692453	2012
GBP-1 acts as a tumor suppressor in colorectal cancer cells. Carcinogenesis\| PubMed ID: 23042300	2013
Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor. Molecular and cellular biology\| PubMed ID: 24190970	2014
Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nature medicine\| PubMed ID: 24784231	2014
Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS. Journal of cellular and molecular medicine\| PubMed ID: 25823874	2015
Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice. Inflammatory bowel diseases\| PubMed ID: 26164664	2015
Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis. International journal of molecular sciences\| PubMed ID: 26861291	2016
Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases. World journal of gastroenterology\| PubMed ID: 27605879	2016

Elisabeth Naschberger

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