The acquisition of a migratory/invasive phenotype by tumor cells is characterized by the loss of cell-cell adhesion contacts and the expression of degradative properties. In this study, we examined ...the effect of the disorganization of occludin/zonula occludens (ZO)-1 tight junction (TJ) complexes on the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP). We first compared the expression of MT1-MMP and the localization of occludin/ZO-1 complexes in breast tumor cell lines displaying various degrees of invasiveness. We showed that the expression of MT1-MMP in invasive breast tumor cell lines correlates with the absence of occludin and with a cytoplasmic localization of ZO-1. In contrast, noninvasive cell lines displayed a membrane staining for both ZO-1 and occludin and did not express MT1-MMP. In vivo, cytoplasmic ZO-1 and MT1-MMP could be detected in invasive tumor clusters of human breast carcinomas. We then used RNA interference strategy to inhibit ZO-1 expression in invasive BT549 cells and to evaluate the effect of ZO-1 down-regulation on MT1-MMP expression. We observed that ZO-1 small interfering RNA transfection down-regulates MT1-MMP mRNAs and proteins and subsequently decreases the ability of tumor cells to invade a reconstituted basement membrane in a Boyden chamber assay. Inversely, transfection of expression vectors encoding wild-type ZO-1 or the NH2-terminal fragment of ZO-1 comprising the PSD95/DLG/ZO-1 domains in BT549 activated a human MT1-MMP promoter luciferase reporter construct and increased cell invasiveness. Such transfections concomitantly activated the beta-catenin/TCF/LEF pathway. Our results therefore show that ZO-1, besides its structural role in TJ assembly, can intervene in signaling events promoting tumor cell invasion.
The formation of multimeric complexes by membrane-type 1 matrix metalloproteinase (MT1-MMP) may facilitate its autocatalytic inactivation or proMMP-2 activation on the cell surface. To characterize ...these processes, we expressed various glutathione S-transferase/MT1-MMP fusion proteins in human HT-1080 fibrosarcoma cells and SV40-transformed lung fibroblasts and analyzed their effects on MT1-MMP activity and potential homophilic interactions. We report here that MT1-MMP is expressed on the cell surface as oligomeric 200–240-kDa complexes containing both the active 60-kDa and autocatalytically processed 43-kDa species. Overexpression of a glutathione S-transferase/MT1-MMP fusion protein containing the transmembrane and cytoplasmic domains of MT1-MMP inhibited the phorbol 12-myristate 13-acetate-induced autocatalytic cleavage of endogenous MT1-MMP to the 43-kDa species, but not proMMP-2 activation. On the other hand, a similar fusion protein with the hemopexin, transmembrane, and cytoplasmic domains inhibited proMMP-2 activation in a dominant-negative fashion. These results suggest that both the autocatalytic cleavage of MT1-MMP and proMMP-2 activation may be regulated by oligomerization through the cytoplasmic and hemopexin domains. Indeed, either domain, when attached to the cell membrane by a transmembrane domain, formed stable homophilic complexes. Copurification of MT1-MMP with these fusion proteins correlated with their cell-surface co-localization. Thus, MT1-MMP oligomerization through the hemopexin, transmembrane, and cytoplasmic domains controls its catalytic activity.
Despite multiple previous studies in the field of vascular anomalies, the mechanism(s) leading to their development, progression and maintenance has remained unclear. In this study, we have ...characterized the expression levels of vascular endothelial growth factors and their receptors in 33 human vascular anomalies. Analysis with quantitative real-time PCR and gene-specific assays showed higher expression of neuropilin-2 (NRP2) and VEGF-receptor-3 (VEGFR-3) mRNAs in vascular malformations (VascM) as compared to infantile hemangiomas (Hem). In addition, the expression levels of PlGF and VEGF-C mRNA were significantly higher in venous VascM when compared to the other VascM and Hem. Higher expression of NRP2 and VEGFR-3 were confirmed by immunohistochemistry. To further study the importance of NRP2 and VEGFR-3, endothelial cell (EC) cultures were established from vascular anomalies. It was found that NRP2 and VEGFR-3 mRNA levels were significantly higher in some of the VascM ECs as compared to human umbilical vein ECs which were used as control cells in the study. Furthermore, adenoviral delivery of soluble decoy NRP2 prevented the proliferation of ECs isolated from most of the vascular anomalies. Our findings suggest that NRP2 functions as a factor maintaining the pathological vascular network in these anomalies. Thus, NRP2 could become a potential therapeutic target for the diagnosis and treatment of vascular anomalies.
Purpose: Pathological vascular differentiation in retinal vein occlusion (RVO)-related neovessel formation remains poorly characterized. The role of intraocular lymphatic-like differentiation or ...endothelial progenitor cell activity has not been studied in this disease. Methods: Vitrectomy was performed in an eye with hemi-RVO; the neovessel membrane located at the optic nerve head was removed and subjected to immunohistochemistry. Characterization of the neovascular tissue was performed using hematoxylin and eosin, α-smooth muscle actin, and the pan-endothelial cell (EC) adhesion molecule CD31. The expression of lymphatic EC markers was studied by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), podoplanin (PDPN), and prospero-related homeobox protein 1 (Prox-1). Potential vascular stem/progenitor cells were identified by active cellular proliferation (Ki67) and expression of the stem cell marker CD117. Results: The specimen contained blood vessels lined by ECs and surrounded by pericytes. Immunoreactivity for LYVE-1 and Prox-1 was detected, with Prox-1 being more widely expressed in the active Ki67-positive lumen-lining cells. PDPN expression was instead found in the cells residing in the extravascular tissue. Expression of the stem cell markers CD117 and Ki67 suggested vascular endothelial progenitor cell activity. Conclusions: Intraocular lymphatic-like differentiation coupled with progenitor cell activation may be involved in the pathology of neovessel formation in ischemia-induced human hemi-RVO.
Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-β1 (TGF-β1) to extracellular matrices and its release and ...activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-β1, its propeptide (β1-latency-associated protein), and latent TGF-β-binding protein and incorporated latent TGF-β1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-β1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-β1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized Mr 25,000 TGF-β1 but did not dissociate high Mr latent TGF-β1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-β1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-β from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.
Diagnostic criteria, progression risk and optimal monitoring for intestinal failure (IF)-associated liver disease (IFALD) remain undefined. We assessed predictors, non-invasive markers and ...progression of histopathological liver disease in patients with IF.
In total, 77 children with IF and median age of 1.7 years underwent diagnostic liver biopsy, which was repeated in 48 patients after 2.9 years with simultaneous evaluation of liver biochemistry, liver stiffness, serum citrulline (a surrogate for viable enterocyte mass), spleen size, esophageal varices and clinical data. Patients were staged according to histopathological liver disease activity: active IFALD (cholestasis and/or inflammation), chronic IFALD (significant fibrosis and/or steatosis), or no IFALD (none of these features).
Diagnostic liver biopsy revealed active, chronic or no IFALD in 48%, 21% and 31% of patients. Active IFALD was segregated by low serum citrulline, parenteral nutrition (PN) dependency and young age, while weaning off PN and older age predicted chronic IFALD. Although the liver histopathology in most patients either normalized (52%) or transformed to a less reactive (chronic) disease stage (23%), 19% of patients retained and 6.3% progressed to an active cholestatic/inflammatory IFALD phenotype. Decreased serum citrulline and PN-dependency also predicted active IFALD in follow-up biopsies. Increased median liver biochemistry values and liver stiffness only associated with active IFALD, which was accurately identified by gamma-glutamyltransferase (GGT), citrulline and liver stiffness, their combinations reaching diagnostic and follow-up AUROC values above 0.90.
Active IFALD, essentially predicted by intestinal disruption and PN-dependency, was accurately detected by GGT, liver stiffness and citrulline, which together with recent advances in clinical management options, provides new avenues for monitoring and targeted liver protection in patients with IF.
Liver disease is a common and critical complication in patients with intestinal failure, who require intravenous nutrition for survival due to severe intestinal dysfunction. We showed that both intravenous nutrition dependency and intestinal disruption essentially predicted development of active histopathological liver disease, which persisted in 25% of patients during long-term follow-up and could be accurately detected without the need for liver biopsy. Identification of the active and potentially progressive histopathology offers new possibilities for monitoring and targeted liver protection in patients with intestinal failure.
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•Histological cholestasis and/or inflammation defined active IFALD.•Of 77 children with intestinal failure, 48% had active IFALD at diagnostic liver biopsy.•25% had retained or progressed to active IFALD in follow-up biopsy after 3 years.•Low citrulline and parenteral nutrition predicted active IFALD at both biopsies.•Citrulline, GGT and liver stiffness accurately diagnosed active IFALD.
OBJECTIVE:Although liver disease is a major complication of parenteral nutrition (PN) for intestinal failure (IF), its pathogenesis remains unclear. We investigated potential molecular mechanisms of ...liver injury in pediatric onset IF.
METHODS:Liver expression of canalicular phospholipid (ABCB4), bile acid (ABCB11), and sterol (ABCG5/8) transporters, their upstream regulators LXR and FXR as well as pro-inflammatory cytokines interleukin-6 (IL6) and tumor necrosis factor (TNF) were investigated among patients with IF age median 3.8 (IQR 1.2 to 11) in relation to biochemical and histologic liver injury, PN, serum plant sterols, fibroblast growth factor 19, and α-tocopherol.
RESULTS:Patients receiving PN currently (n = 18) showed more advanced liver injury than patients after weaning off PN (n = 30). Histologic portal inflammation strongly segregated PN-dependent (44%) from weaned off patients (3%, P = 0.001) and coupled with progression of cholestasis and liver fibrosis. Patients with portal inflammation demonstrated markedly induced liver RNA expression of IL6 and TNF, repression of FXR and its canalicular bile transporter target gene RNA expression, including ABCB4 and ABCB11 as well as decreased protein expression of ABCB11 and ABCB4. Furthermore, upregulation of LXR and ABCG5/8 RNA expression was suppressed in patients with portal inflammation. Current PN, increased serum levels of plant sterols stigmasterol, avenasterol, and sitosterol along with serum citrulline, a marker of enterocyte mass, predicted portal inflammation.
CONCLUSIONS:In pediatric onset IF, current PN delivery synergistically with intestinal compromise promote liver inflammation, which associates with progression of biochemical and histologic liver injury, while reducing expression of canalicular bile transporters.
Childhood interstitial lung disease (chILD) is an umbrella concept covering a wide range of rare lung diseases, many of which are unique to childhood. The diagnosis is based on clinical presentation, ...multidetector computed tomography (MDCT), genetic testing, lung-function testing, and lung biopsy. Because knowledge of the usefulness of MDCT pattern recognition in ChILD is at present limited, we examined the occurrence of MDCT patterns in children with histologically confirmed interstitial lung disease.
We searched the biopsy, MDCT, and clinical information database of a single national paediatric referral hospital for 2004–2020. Data were from affected children under age 18. MDCT images we reanalysed while blinded to the identity and referral information.
We included 90 patients, of whom 63 (70 %) were male. The median age at biopsy was 1.3 years (interquartile range 0.1–16.8). Biopsy findings fell into 26 histological classes covering all nine chILD classification categories. We recognized six distinct MDCT patterns: neuroendocrine cell hyperplasia of infancy (23), organizing pneumonia (5), non-specific interstitial pneumonia (4), bronchiolitis obliterans (3), pulmonary alveolar proteinosis (2), and bronchopulmonary dysplasia (n = 2). Of the total 90, in 51 (57 %) children, none of these six MDCT patterns appeared. Of those 39 children with a recognizable MDCT pattern, in 34 (87 %), that pattern predicted their final diagnosis.
Among cases of chILD, we identified a specific predefined MDCT pattern in only 43 %. However, when such a recognizable pattern occurred, it was predictive of the final chILD diagnosis.
To evaluate the association between Wilms tumor histology at diagnosis and the change in Wilms' tumor volume during preoperative chemotherapy.
We included all the 52 patients operated for Wilms tumor ...at 1988–2015, who had both pathology samples and either CT or MRI-images before and after preoperative chemotherapy, available for reevaluation.
The median tumor volume was 586 ml (IQR 323–903) at diagnosis. The median change in tumor volume was −68% (IQR −85 to −40, p < 0.001) and the proportion of tumor necrosis 85% (IQR 24–97), after preoperative chemotherapy. There was a correlation between blastemal cell content in prechemotherapy cutting needle biopsy (CNB) sample and the reduction in tumor volume (Rho = −0.452, p = 0.002). High stromal and epithelial cell contents in CNB samples were associated with the lesser change in tumor volume (Rho = 0.279, p =0.053 and Rho = 0.300, p = 0.038 respectively). Reduction of tumor volume and the proportion of tumor necrosis after chemotherapy were associated (Rho = −0.502, p < 0.001). The actual viable tumor volume decreased in median by 97% (IQR 65–100), and the decrease could be seen in all cellular components. In three patients, the tumor volume increased more than 10% during the preoperative chemotherapy. Two of them had anaplastic tumor in the nephrectomy specimen.
Wilms tumor total and viable tumor volumes were reduced by 68% and 97% with preoperative chemotherapy, respectively. High proportion of blastemal cells in CNB was associated with greatest decrease in Wilms tumor volume. Increase in tumor volume during preoperative chemotherapy may indicate anaplastic tumor and prolonging of preoperative therapy should be avoided.
Retrospective review.
Level III.
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