| 1. |
- Chang, Ya-Ting, et al.
(author)
-
Versican accumulates in vascular lesions in pulmonary arterial hypertension
- 2016
-
In: PULMONARY CIRCULATION. - : Wiley. - 2045-8932 .- 2045-8940. ; 6:3, s. 347-359
-
Journal article (peer-reviewed)abstract
- Pulmonary arterial hypertension (PAH) is a lethal condition for which there is no effective curative pharmacotherapy. PAH is characterized by vasoconstriction, wall thickening of pulmonary arteries, and increased vascular resistance. Versican is a chondroitin sulfate proteoglycan in the vascular extracellular matrix that accumulates following vascular injury and promotes smooth-muscle cell proliferation in systemic arteries. Here, we investigated whether versican may play a similar role in PAH. Paraffin-embedded lung sections from patients who underwent lung transplantation to treat PAH were used for immunohistochemistry. The etiologies of PAH in the subjects involved in this study were idiopathic PAH, scleroderma, and congenital heart disease (atrial septal defect) with left-to-right shunt. Independent of the underlying etiology, increased versican immunostaining was observed in areas of medial thickening, in neointima, and in plexiform lesions. Western blot of lung tissue lysates confirmed accumulation of versican in patients with PAH. Double staining for versican and CD45 showed only occasional colocalization in neointima of high-grade lesions and plexiform lesions. In vitro, metabolic labeling with [S-35] sulfate showed that human pulmonary artery smooth-muscle cells (hPASMCs) produce mainly chondroitin sulfate glycosaminoglycans. In addition, hypoxia, but not cyclic stretch, was demonstrated to increase both versican messenger RNA expression and protein synthesis by hPASMCs. Versican accumulates in vascular lesions of PAH, and the amount of versican correlates more with lesion severity than with underlying etiology or inflammation. Hypoxia is a possible regulator of versican accumulation, which may promote proliferation of pulmonary smooth-muscle cells and vascular remodeling in PAH.
|
|
| 2. |
- Hildebrand, S, et al.
(author)
-
The E-cadherin/AmotL2 complex organizes actin filaments required for epithelial hexagonal packing and blastocyst hatching
- 2017
-
In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1, s. 9540-
-
Journal article (peer-reviewed)abstract
- Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.
|
|
| 3. |
- Retamal, Jaime, et al.
(author)
-
High respiratory rate is associated with early reduction of lung edema clearance in an experimental model of ARDS
- 2016
-
In: Acta Anaesthesiologica Scandinavica. - : Wiley. - 0001-5172 .- 1399-6576. ; 60:1, s. 79-92
-
Journal article (peer-reviewed)abstract
- BACKGROUND: The independent impact of respiratory rate on ventilator-induced lung injury has not been fully elucidated. The aim of this study was to investigate the effects of two clinically relevant respiratory rates on early ventilator-induced lung injury evolution and lung edema during the protective ARDSNet strategy. We hypothesized that the use of a higher respiratory rate during a protective ARDSNet ventilation strategy increases lung inflammation and, in addition, lung edema associated to strain-induced activation of transforming growth factor beta (TGF-β) in the lung epithelium.METHODS: Twelve healthy piglets were submitted to a two-hit lung injury model and randomized into two groups: LRR (20 breaths/min) and HRR (40 breaths/min). They were mechanically ventilated during 6 h according to the ARDSNet strategy. We assessed respiratory mechanics, hemodynamics, and extravascular lung water (EVLW). At the end of the experiment, the lungs were excised and wet/dry ratio, TGF-β pathway markers, regional histology, and cytokines were evaluated.RESULTS: No differences in oxygenation, PaCO2 levels, systemic and pulmonary arterial pressures were observed during the study. Respiratory system compliance and mean airway pressure were lower in LRR group. A decrease in EVLW over time occurred only in the LRR group (P < 0.05). Wet/dry ratio was higher in the HRR group (P < 0.05), as well as TGF-β pathway activation. Histological findings suggestive of inflammation and inflammatory tissue cytokines were higher in LRR.CONCLUSION: HRR was associated with more pulmonary edema and higher activation of the TGF-β pathway. In contrast with our hypothesis, HRR was associated with less lung inflammation.
|
|
| 4. |
- Schwochow, Doreen, et al.
(author)
-
The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A
- 2017
-
In: PLOS Genetics. - : PUBLIC LIBRARY SCIENCE. - 1553-7390 .- 1553-7404. ; 13:4
-
Journal article (peer-reviewed)abstract
- Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.
|
|
| 5. |
- Veerman, Rosanne E., et al.
(author)
-
Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin
- 2021
-
In: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 10:9
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 mu l or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.
|
|
| 6. |
- Wu, Chengjun, et al.
(author)
-
HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice
- 2016
-
In: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 489, s. 44-50
-
Journal article (peer-reviewed)abstract
- Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver.
|
|
| 7. |
- Younis, Shady, et al.
(author)
-
The importance of the ZBED6-IGF2 axis for metabolic regulation in mouse myoblast cells
- 2020
-
In: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 34:8, s. 10250-10266
-
Journal article (peer-reviewed)abstract
- The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5'-GGCTCG-3' in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT ) or complete ablation of Zbed6 leads to ~20-fold upregulation of the IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 upregulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of upregulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities, and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study adds new insights how the ZBED6-Igf2 axis affects muscle metabolism.
|
|
