| 1. |
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| 2. |
- Ajalloueian, Fatemeh, et al.
(författare)
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Investigation of Human Mesenchymal Stromal Cells Cultured on PLGA orPLGA/Chitosan Electrospun Nanofibers
- 2015
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Ingår i: Journal of Bioprocessing & Biotechniques. - : OMICS Publishing Group. - 2155-9821. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- We compared the viability, proliferation, and differentiation of human Mesenchymal Stromal Cells (MSC)after culture on poly(lactic-co-glycolic acid) (PLGA) and PLGA/chitosan (PLGA/CH) hybrid scaffolds. We appliedconventional and emulsion electrospinning techniques, respectively, for the fabrication of the PLGA and PLGA/CH scaffolds. Electrospinning under optimum conditions resulted in an average fiber diameter of 166 ± 33 nmfor the PLGA/CH and 680 ± 175 nm for the PLGA scaffold. The difference between the tensile strength of thePLGA and PLGA/CH nanofibers was not significant, but PLGA/CH showed a significantly lower tensile modulusand elongation at break. However, it should be noted that the extensibility of the PLGA/CH was higher than thatof the nanofibrous scaffolds of pure chitosan. As expected, a higher degree of hydrophilicity was seen with PLGA/CH, as compared to PLGA alone. The biocompatibility of the PLGA and PLGA/CH scaffolds was compared usingMTS assay as well as analysis by scanning electron microscopy and confocal microscopy. The results showed thatboth scaffold types supported the viability and proliferation of human MSC, with significantly higher rates on PLGA/CH nanofibers. Nonetheless, an analysis of gene expression of MSC grown on either PLGA or PLGA/CH showed asimilar differentiation pattern towards bone, nerve and adipose tissues.
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| 3. |
- Asif, Sana, et al.
(författare)
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Oxygen-charged HTK-F6H8 emulsion reduces ischemia : reperfusion injury in kidneys from brain-dead pigs
- 2012
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Ingår i: Journal of Surgical Research. - : Elsevier BV. - 0022-4804 .- 1095-8673. ; 178:2, s. 959-967
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Tidskriftsartikel (refereegranskat)abstract
- Background:Prolonged cold ischemia is frequently associated with a greater risk of delayed graft function and enhanced graft failure. We hypothesized that media, combining a high oxygen-dissolving capacity with specific qualities of organ preservation solutions, would be more efficient in reducing immediate ischemia-reperfusion injury from organs stored long term compared with standard preservation media.Methods:Kidneys retrieved from brain-dead pigs were flushed using either cold histidine-tryptophan-ketoglutarate (HTK) or oxygen-precharged emulsion composed of 75% HTK and 25% perfluorohexyloctane. After 18 h of cold ischemia the kidneys were transplanted into allogeneic recipients and assessed for adenosine triphosphate content, morphology, and expression of genes related to hypoxia, environmental stress, inflammation, and apoptosis.Results:Compared with HTK-flushed kidneys, organs preserved using oxygen-precharged HTK-perfluorohexyloctane emulsion had increased elevated adenosine triphosphate content and a significantly lower gene expression of hypoxia inducible factor-1 alpha, vascular endothelial growth factor, interleukin-1 alpha, tumor necrosis factor-alpha, interferon-alpha, JNK-1, p38, cytochrome-c, Bax, caspase-8, and caspase-3 at all time points assessed. In contrast, the mRNA expression of Bcl-2 was significantly increased.Conclusions:The present study has demonstrated that in brain-dead pigs the perfusion of kidneys with oxygen-precharged HTK-perfluorohexyloctane emulsion results in significantly reduced inflammation, hypoxic injury, and apoptosis and cellular integrity and energy content are well maintained. Histologic examination revealed less tubular, vascular, and glomerular changes in the emulsion-perfused tissue compared with the HTK-perfused counterparts. The concept of perfusing organs with oxygen-precharged emulsion based on organ preservation media represents an efficient alternative for improved organ preservation.
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| 4. |
- Babiker, Adil A., et al.
(författare)
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Mapping pro- and antiangiogenic factors on the surface of prostasomes of normal and malignant cell origin
- 2010
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Ingår i: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 70:8, s. 834-847
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. Tumor growth is angiogenesis-dependent and the formation of new blood vessels is associated with the increased expression of angiogenic factors. Prostasomes are secretory granules produced, stored and released by the glandular epithelial cells of the prostate. We investigated the expression of selected angiogenic and anti-angiogenic factors on the surface of prostasomes of different origins as well as the direct effect of prostasomes on angiogenesis. METHODS: VEGF, endothelin-1, endostatin, and thrombospondin-1 were determined on prostasomes from seminal fluid and human prostate cancer cell lines (DU145,PC-3,LNCaP) using different immunochemical techniques. Human dermal microvascular endothelial cells were incubated with seminal and DU145 cell-prostasomes and with radioactive thymidine. The effect of prostasomes on angiogenesis was judged by measuring the uptake of labeled thymidine. The presence of any deleterious effects of prostasomes on the endothelial cells was investigated using thymidine assay and confocal laser microscopy. RESULTS: VEGF and endothelin-1 were determined on malignant cell-prostasomes (no difference between cell lines) but not determined on seminal prostasomes. The same applies for the expression of endostatin but with much higher expression on malignant cell-prostasomes with obvious differences between them. Seminal and DU145 cell-prostasomes were found to have anti-angiogenic effect which was more expressed by DU145 cell-prostasomes. No deleterious effect of prostasomes on endothelial function was detected using either thymidine assay or microscopy. CONCLUSIONS: Prostasomes contain pro- and anti-angiogenic factors that function to counteract each other unless the impact from one side exceeds the other to bring about dysequilibrium.
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| 5. |
- Bekkhus, Tove, et al.
(författare)
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Stromal transdifferentiation drives lipomatosis and induces extensive vascular remodeling in the aging human lymph node
- 2023
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Ingår i: Journal of Pathology. - : John Wiley & Sons. - 0022-3417 .- 1096-9896. ; 259:3, s. 236-253
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Tidskriftsartikel (refereegranskat)abstract
- Lymph node (LN) lipomatosis is a common, but rarely discussed phenomenon, associated with aging, involving a gradual exchange of the LN parenchyma into adipose tissue. The mechanisms behind these changes and the effects on the LN are unknown. We show that LN lipomatosis starts in the medullary regions of the human LN and link the initiation of lipomatosis to transdifferentiation of LN fibroblasts into adipocytes. The latter is associated with a downregulation of lymphotoxin beta expression. We also show that, isolated medullary and CD34+ fibroblast, in contrast to the reticular cells of the T-cell zone, display an inherent higher sensitivity for adipogenesis. Progression of lipomatosis leads to a gradual loss of the medullary lymphatic network, but at later stages, collecting-like lymphatic vessels, are found inside the adipose tissue. The stromal dysregulation includes a dramatic remodeling and dilation of the high endothelial venules associated with reduced density of naïve T-cells. Abnormal clustering of plasma cells is also observed. Thus, LN lipomatosis causes widespread stromal dysfunction with consequences for the immune contexture of the human LN. Our data warrant an increased awareness of LN lipomatosis as a factor contributing to decreased immune functions in the elderly and in disease.
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| 6. |
- Berg, Aase, et al.
(författare)
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Complement Activation Correlates With Disease Severity and Contributes to Cytokine Responses in Plasmodium falciparum Malaria
- 2015
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Ingår i: The Internet Journal of Infectious Diseases. - : Oxford University Press (OUP). - 1528-8366 .- 0022-1899 .- 1537-6613. ; 212:11, s. 1835-1840
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Tidskriftsartikel (refereegranskat)abstract
- The impact of complement activation and its possible relation to cytokine responses during malaria pathology was investigated in plasma samples from patients with confirmed Plasmodium falciparum malaria and in human whole-blood specimens stimulated with malaria-relevant agents ex vivo. Complement was significantly activated in the malaria cohort, compared with healthy controls, and was positively correlated with disease severity and with certain cytokines, in particular interleukin 8 (IL-8)/CXCL8. This was confirmed in ex vivo-stimulated blood specimens, in which complement inhibition significantly reduced IL-8/CXCL8 release. P. falciparum malaria is associated with systemic complement activation and complement-dependent release of inflammatory cytokines, of which IL-8/CXCL8 is particularly prominent.
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| 7. |
- Beter, M., et al.
(författare)
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SproutAngio : an open-source bioimage informatics tool for quantitative analysis of sprouting angiogenesis and lumen space
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional image analyses are required to improve the understanding of the regulation of blood vessel formation and heterogeneity. Currently, quantitation of 3D endothelial structures or vessel branches is often based on 2D projections of the images losing their volumetric information. Here, we developed SproutAngio, a Python-based open-source tool, for fully automated 3D segmentation and analysis of endothelial lumen space and sprout morphology. To test the SproutAngio, we produced a publicly available in vitro fibrin bead assay dataset with a gradually increasing VEGF-A concentration (). We demonstrate that our automated segmentation and sprout morphology analysis, including sprout number, length, and nuclei number, outperform the widely used ImageJ plugin. We also show that SproutAngio allows a more detailed and automated analysis of the mouse retinal vasculature in comparison to the commonly used radial expansion measurement. In addition, we provide two novel methods for automated analysis of endothelial lumen space: (1) width measurement from tip, stalk and root segments of the sprouts and (2) paired nuclei distance analysis. We show that these automated methods provided important additional information on the endothelial cell organization in the sprouts. The pipelines and source code of SproutAngio are publicly available ().
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| 8. |
- Bongoni, Anjan K., et al.
(författare)
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Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno) transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO. hCD46. hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFa-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO. hCD46. hTBM PAEC. Coating of untreated or hTNFa-treated PAEC with CHC (100 mu g/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 +/- 16.1 min, p < 0.001) compared to WT PAEC (34.0 +/- 8.2 min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3 +/- 11.3 min, p < 0.001; GTKO. hCD46. hTBM: 146.2 +/- 20.4 min, p < 0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.
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| 9. |
- Cabric, Sanja, et al.
(författare)
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Anchoring of vascular endothelial growth factor to surface-immobilized heparin on pancreatic islets : implications for stimulating islet angiogenesis
- 2010
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Ingår i: Tissue engineering. Part A. - : Mary Ann Liebert Inc. - 1937-3341 .- 1937-335X. ; 16:3, s. 961-970
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Tidskriftsartikel (refereegranskat)abstract
- In pancreatic islet transplantation, early revascularization is necessary for long-term graft function. We have shown in in vitro and in vivo models that modification with surface-attached heparin protects the islets from acute attack by the innate immune system of the blood following intraportal islet transplantation. In this study, we have investigated the ability of an immobilized conjugate composed of heparin to bind the angiogenic growth factor vascular endothelial growth factor-A (VEGF-A) as a means of attracting endothelial cells (ECs) to induce angiogenesis and revascularization. We analyzed the capacity of VEGF-A to bind to immobilized heparin and how this affected the proliferation and adherence of ECs to both artificial glass surfaces and islets. Quartz crystal microbalance with dissipation monitoring and slot-blot demonstrated the binding of VEGF-A to heparin-coated surfaces upon which ECs showed protein-dependent proliferation. Also, ECs cultured on heparin-coated glass surfaces exhibited effects upon focal contacts. Heparinized islets combined with VEGF-A demonstrated unaffected insulin release. Further, covering islets with heparin also increased the adhesion of ECs to the islet surface. Immobilized heparin on the islet surface may be a useful anchor molecule for achieving complete coverage of islets with angiogenic growth factors, ultimately improving islet revascularization and engraftment in pancreatic islet transplantation.
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| 10. |
- Cabric, Sanja, 1977-
(författare)
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Pancreatic Islet Transplantation : Modifications of Islet Properties to Improve Graft Survival
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization. Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process. The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.
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| 11. |
- Corada, Monica, et al.
(författare)
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Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier
- 2019
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Ingår i: Circulation Research. - 0009-7330 .- 1524-4571. ; 124:4, s. 511-525
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Tidskriftsartikel (refereegranskat)abstract
- Rationale: The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/β-catenin signaling is responsible for the early phases of brain vascularization and BBB differentiation. However, this signal declines after birth, and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown.Objective: Sox17 (SRY [sex-determining region Y]-box 17) constitutes a major downstream target of Wnt/β-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/β-catenin in inducing BBB differentiation and maintenance.Methods and Results: Using reporter mice and nuclear staining of Sox17 and β-catenin, we report that although β-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo and progressively declines after birth. In search of Sox17 mechanism of action, RNA sequencing analysis of gene expression of brain endothelial cells has identified members of the Wnt/β-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/β-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the β-catenin destruction complex or expression of a degradation-resistant β-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17.Conclusions: Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB, and they underline the strict reciprocal tuning of this transcription factor and Wnt/β-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.
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| 12. |
- Cottarelli, Azzurra, et al.
(författare)
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Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/beta-catenin signaling
- 2020
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Ingår i: Development. - : COMPANY BIOLOGISTS LTD. - 0950-1991 .- 1477-9129. ; 147:16
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Tidskriftsartikel (refereegranskat)abstract
- Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/beta-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/beta-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/beta-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNSregions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/beta-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro. Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/beta-catenin activity.
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| 13. |
- Cross, Michael J, et al.
(författare)
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The Shb Adaptor Protein Binds to Tyrosine 766 in the FGFR-1 and Regulatesthe Ras/MEK/MAPK Pathway via FRS2 Phosphorylation in Endothelial Cells
- 2002
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 13:8, s. 2881-2893
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Tidskriftsartikel (refereegranskat)abstract
- Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-γ (PLC-γ). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-α and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-γ activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1–mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-γ activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1–mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1–mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.
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| 14. |
- Cunha, Sara I., et al.
(författare)
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Deregulated TGF-beta/BMP Signaling in Vascular Malformations
- 2017
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Ingår i: Circulation Research. - 0009-7330 .- 1524-4571. ; 121:8, s. 981-999
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Forskningsöversikt (refereegranskat)abstract
- Correct organization of the vascular tree requires the balanced activities of several signaling pathways that regulate tubulogenesis and vascular branching, elongation, and pruning. When this balance is lost, the vessels can be malformed and fragile, and they can lose arteriovenous differentiation. In this review, we concentrate on the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) pathway, which is one of the most important and complex signaling systems in vascular development. Inactivation of these pathways can lead to altered vascular organization in the embryo. In addition, many vascular malformations are related to deregulation of TGF-beta/BMP signaling. Here, we focus on two of the most studied vascular malformations that are induced by deregulation of TGF-beta/BMP signaling: hereditary hemorrhagic telangiectasia (HHT) and cerebral cavernous malformation (CCM). The first of these is related to loss-of-function mutation of the TGF-beta/BMP receptor complex and the second to increased signaling sensitivity to TGF-beta/BMP. In this review, we discuss the potential therapeutic targets against these vascular malformations identified so far, as well as their basis in general mechanisms of vascular development and stability.
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| 15. |
- Duprez, Ida Rasmusson, et al.
(författare)
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Preparatory studies of composite mesenchymal stem cell islets for application in intraportal islet transplantation
- 2011
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Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 116:1, s. 8-17
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Background. Low engraftment and adverse immune reactions hamper the success rate of clinical islet transplantation. In this study, we investigated the capacity of human mesenchymal stem cells (MSCs) to adhere to human islets of Langerhans and their effects in immune modulation and during blood interactions in vitro. Methods. Composite MSC-islets were formed by suspension co-culture, and the phenotype was evaluated by confocal microscopy. Islet function was assessed by dynamic insulin release in response to glucose in vitro. Mixed lymphocyte-islet reactions (MLIR) and the tubing blood loop model were utilized as in vitro tools to analyse the effect of MSCs on the innate and adaptive immune reactions triggered by the islets. Results. MSCs rapidly adhered to islets and spread out to cover the islet surface. Insulin expression and secretion were sustained with the MSC coating. MSC-coated islets showed unaffected reactions with blood in vitro in comparison to control islets. Furthermore, MSCs suppressed lymphocyte proliferation induced by islet cells in MLIR. Conclusion. We conclude that it is possible to create composite MSC-islets to enable delivery of the MSCs by utilizing the adhesive capacity of the MSCs. This could have beneficial immunosuppressive effects in optimizing pancreatic islet transplantation.
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| 16. |
- Edin, Fredrik, et al.
(författare)
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3-D gel culture and time-lapse video microscopy of the human vestibular nerve
- 2014
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Ingår i: Acta Oto-Laryngologica. - : Informa UK Limited. - 0001-6489 .- 1651-2251. ; 134:12, s. 1211-1218
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Tidskriftsartikel (refereegranskat)abstract
- UNLABELLED: Abstract Conclusions: Human inner ear neurons have an innate regenerative capacity and can be cultured in vitro in a 3-D gel. The culture technique is valuable for experimental investigations of human inner ear neuron signaling and regeneration.OBJECTIVES: To establish a new in vitro model to study human inner ear nerve signaling and regeneration.METHODS: Human superior vestibular ganglion (SVG) was harvested during translabyrinthine surgery for removal of vestibular schwannoma. After dissection tissue explants were embedded and cultured in a laminin-based 3-D matrix (Matrigel™). 3-D growth cone (GC) expansion was analyzed using time-lapse video microscopy (TLVM). Neural marker expression was appraised using immunocytochemistry with fluorescence and laser confocal microscopy.RESULTS: Tissue explants from adult human SVG could be cultured in 3-D in a gel, indicating an innate potential for regeneration. Cultured GCs were found to expand dynamically in the gel. Growth cone expansion and axonal Schwann cell alignment were documented using TLVM. Neurons were identified morphologically and through immunohistochemical staining.
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| 17. |
- Edin, Fredrik, et al.
(författare)
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Differentiation of human neural progenitor cell-derived spiral ganglion-like neurons : a time-lapse video study
- 2014
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Ingår i: Acta Oto-Laryngologica. - : Informa UK Limited. - 0001-6489 .- 1651-2251. ; 134:5, s. 441-447
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Tidskriftsartikel (refereegranskat)abstract
- Conclusions: Human neural progenitor cells can differentiate into spiral ganglion-like cells when exposed to inner ear-associated growth factors. The phenotype bears resemblance to human sphere-derived neurons. Objective: To establish an in vitro model for the human auditory nerve to replace and complement in vivo animal experiments and ultimately human in vivo transplantation. Methods: Human neural progenitors were differentiated under conditions developed for in vitro survival of human primary spiral ganglion culture with media containing growth factors associated with inner ear development. Differentiation was documented using time-lapse video microscopy. Time-dependent marker expression was evaluated using immunocytochemistry with fluorescence and laser confocal microscopy. Results: Within 14 days of differentiation, neural progenitors adopted neural phenotype and expressed spiral ganglion-associated markers.
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| 18. |
- Edin, Fredrik, 1985-
(författare)
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Strategies in Cochlear Nerve Regeneration, Guidance and Protection : Prospects for Future Cochlear Implants
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Today, it is possible to restore hearing in congenitally deaf children and severely hearing-impaired adults through cochlear implants (CIs). A CI consists of an external sound processor that provides acoustically induced signals to an internal receiver. The receiver feeds information to an electrode array inserted into the fluid-filled cochlea, where it provides direct electrical stimulation to the auditory nerve. Despite its great success, there is still room for improvement, so as to provide the patient with better frequency resolution, pitch information for music and speech perception and overall improved quality of sound. A better stimulation mode for the auditory nerves by increasing the number of stimulation points is believed to be a part of the solution. Current technology depends on strong electrical pulses to overcome the anatomical gap between neurons and the CI. The spreading of currents limits the number of stimulation points due to signal overlap and crosstalk.Closing the anatomical gap between spiral ganglion neurons and the CI could lower the stimulation thresholds, reduce current spread, and generate a more discrete stimulation of individual neurons. This strategy may depend on the regenerative capacity of auditory neurons, and the ability to attract and guide them to the electrode and bridge the gap.Here, we investigated the potential of cultured human and murine neurons from primary inner ear tissue and human neural progenitor cells to traverse this gap through an extracellular matrix gel.Furthermore, nanoparticles were used as reservoirs for neural attractants and applied to CI electrode surfaces. The nanoparticles retained growth factors, and inner ear neurons showed affinity for the reservoirs in vitro.The potential to obtain a more ordered neural growth on a patterned, electrically conducting nanocrystalline diamond surface was also examined. Successful growth of auditory neurons that attached and grew on the patterned substrate was observed.By combining the patterned diamond surfaces with nanoparticle-based reservoirs and nerve-stimulating gels, a novel, high resolution CI may be created. This strategy could potentially enable the use of hundreds of stimulation points compared to the 12 – 22 used today. This could greatly improve the hearing sensation for many CI recipients.
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| 19. |
- Fransson, Moa, et al.
(författare)
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CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery
- 2012
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Ingår i: Journal of Neuroinflammation. - : Springer Science and Business Media LLC. - 1742-2094. ; 9, s. 112-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naive CD4 cells and demonstrate their efficacy in the EAE model.METHODSCD4+T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.RESULTSThe engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptomfree mice were echallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.CONCLUSIONCNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.
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| 20. |
- Fransson, Moa, et al.
(författare)
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Intranasal Delivery of CNS-Retargeted Human Mesenchymal Stromal Cells Prolongs Treatment Efficacy of Experimental Autoimmune Encephalomyelitis
- 2014
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Ingår i: Immunology. - : Wiley. - 0019-2805 .- 1365-2567. ; 142:3, s. 431-441
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Tidskriftsartikel (refereegranskat)abstract
- Treatment with mesenchymal stromal cells (MSC) is currently of interest for a number of diseases including multiple sclerosis (MS). MSCs is well known to target inflamed tissues however, in a therapeutic scenery, systemic administration will lead to few cells reaching the brain. We hypothesized that MSCs may target the brain upon intranasal (i.n) administration and persist in CNS tissue if expressing a CNS-targeting receptor. To demonstrate proof of concept, MSCs were genetically engineered to express a myelin oligodendrocyte glycoprotein (MOG)-specific receptor. Engineered MSCs retained their immunosuppressive capacity, infiltrated into the brain upon i.n. cell administration, and were able to significantly reduce disease symptoms of experimental autoimmune encephalomyelitis (EAE). The mice treated with CNS-targeting MSCs were resistant to further EAE induction whereas non-targeted MSC did not give such persistent effects. Histological analysis revealed increased brain restoration in engineered MSC-treated mice. In conclusion, MSCs can be genetically engineered to target the brain and prolong therapeutic efficacy in an EAE model.
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| 21. |
- Fransson, Moa, et al.
(författare)
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Mesenchymal stromal cells supportendothelial cell interactions in anintramuscular islet transplantation model
- 2015
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Ingår i: Regenerative Medicine Research. - : Springer Science and Business Media LLC. - 2050-490X. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Background:Mesenchymal stromal cells (MSC) have been under investigation for a number of therapies andhave lately been in focus as immunosuppressive actors in the field of transplantation. Herein we haveextended our previously published in vitro model of MSC-islets in an experimental setting of islettransplantation to the abdominal muscle.Human islets coated with luciferase-GFP transduced human MSC were transplanted to the abdomen muscletissue of NOD-scid ILR2γnull mice and cellular interactions were investigated by confocal microscopy.Results:The MSC reduced fibrotic encapsulation and facilitated endothelial cell interactions. In particular, weshow a decreased fraction of αSMA expressing fibrotic tissue surrounding the graft in presence of MSC-isletscompared to islets solely distributed into the muscle tissue. Also, in the presence of MSC, human isletendothelial cells migrated from the center of the graft out into the surrounding tissue forming chimeric bloodvessels with recipient endothelial cells. Further, in the graft periphery, MSC were seen interacting with infiltratingmacrophages.Conclusions:Here, in our experimental in vivo model of composite human islets and luciferase-GFP-transducedhuman MSC, we enable the visualization of close interactions between the MSC and the surrounding tissue. In thismodel of transplantation the MSC contribute to reduced fibrosis and increased islet endothelial cell migration.Furthermore, the MSC interact with the recipient vasculature and infiltrating macrophages.
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| 22. |
- Globisch, Maria Ascencion, et al.
(författare)
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Dysregulated Hemostasis and Immunothrombosis in Cerebral Cavernous Malformations
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:20
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Forskningsöversikt (refereegranskat)abstract
- Cerebral cavernous malformation (CCM) is a neurovascular disease that affects 0.5% of the general population. For a long time, CCM research focused on genetic mutations, endothelial junctions and proliferation, but recently, transcriptome and proteome studies have revealed that the hemostatic system and neuroinflammation play a crucial role in the development and severity of cavernomas, with some of these publications coming from our group. The aim of this review is to give an overview of the latest molecular insights into the interaction between CCM-deficient endothelial cells with blood components and the neurovascular unit. Specifically, we underscore how endothelial dysfunction can result in dysregulated hemostasis, bleeding, hypoxia and neurological symptoms. We conducted a thorough review of the literature and found a field that is increasingly poised to regard CCM as a hemostatic disease, which may have implications for therapy.
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| 23. |
- Globisch, Maria A., et al.
(författare)
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Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation
- 2022
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 140:20, s. 2154-2169
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Tidskriftsartikel (refereegranskat)abstract
- Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.
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| 24. |
- Globisch, Maria Ascención
(författare)
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Inflammation and immunothrombosis in cerebral cavernous malformation : Novel molecular targets for the treatment of an incurable disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cerebral cavernous malformation (CCM) is a vascular disease that causes mulberry-like lesions (cavernomas) in the central nervous system (CNS). Cavernomas are fragile, leaky and prone to rupture which may cause symptoms such as epileptic seizures, focal neurological deficits and hemorrhagic strokes. CCM lesions can appear sporadically in 0.5% of the general population. Alternatively, CCM lesions appear as a consequence of a loss-of-function germline mutation in either CCM1, CCM2, or CCM3 in endothelial cells of the CNS. Inherited CCM is termed familial, and it affects approximately one in ten thousand individuals in an autosomal dominant manner. The aim of this thesis project is to identify novel cell and molecular mechanisms that contribute to the development and progression of cavernous malformations. Additionally, this thesis project aims to identify and validate inhibitors that may reduce lesions and alleviate the side effects in CCM. In this thesis project, two inducible endothelial specific Ccm3 deficient mouse models (acute and chronic) were evaluated with methods such as RNA-sequencing, immunofluorescence, ELISA, scanning and transmission electron microscopy, flow cytometry, and in situ hybridization. Moreover, in vitro cell cultures were used with methods such as immunofluorescence, qPCR, and western blot. Importantly, sporadic and familial human CCM samples were used to show the clinical relevance of our studies. In paper I we focused on the role and kinetics of inflammation in CCM. We analyzed the transcriptome of healthy and Ccm3 deficient mouse brain endothelial cells and found that genes related to inflammation were upregulated in CCM pathology. We identified various inflammatory cytokines in vivo and also identified neutrophils as the most prominent immune cell subtype in CCM. Moreover, we found that neutrophils in CCM produce neutrophil extracellular traps (NETs), that can be inhibited with DNase I. The inhibition of NETs stabilized cavernoma vasculature by reducing fibrinogen and IgG leakage. In paper II we re-used the acute RNA-seq database from paper 1 and focused on endothelial hemostasis and hypoxia. We found that genes related to procoagulation, anticoagulation, and hypoxia were highly upregulated in Ccm3 deficient mice. We validated the findings in vivo and found that the hemostatic system in CCM is dysregulated and that it causes, bleeding, thrombosis, and cerebral hypoxia. In paper III we evaluated the effect of propranolol in CCM. We treated chronic CCM mice with the beta-blocker propranolol and found that propranolol was able to reduce lesions in the brains and retinas of CCM mice as well as reduce cadaverine leakage. Importantly, we identified endothelial plasmalemmal pits and a thick basal membrane between endothelial cells and pericytes, pathological features which reduced upon propranolol treatment. Altogether this thesis significantly contributes to the CCM field as it identified pathological features of cavernomas such as neutrophils with NETs, endothelial plasmalemmal pits, and polyhedrocytes. This thesis work also evaluated pharmacological inhibitors (DNase I and propranolol) in mouse models of CCM and supports the use of anticoagulant therapies in patients with CCM.
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| 25. |
- Herre, Melanie, 1991-
(författare)
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Neutrophil extracellular traps as potential therapeutic targets to prevent tumor-induced organ failure and metastasis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer does not only affect surrounding tissues, but also leads to complications at distant sites. The reason for this is that tumors secrete factors and prime cells that travel through the body, making cancer a systemic disease. In fact, cancer death is mostly caused by systemic complications such as metastasis, organ failure or thrombosis. However, surprisingly little is known about the effect of cancer on distant organs that are not sites of tumor growth.Neutrophil extracellular traps (NETs) are web-like structures consisting of neutrophil DNA and granular proteins that are released from the neutrophil in response to a stimulus. NETs are not only formed in bacterial infections but also in noninfectious inflammatory disorders like cancer. While NETs can be beneficial for the host in severe infections they come with a cost. NETs are pro-thrombotic, cause damage to the vessel-lining endothelial cells, are involved in metastasis formation and impair organ function. The removal of NETs by DNase I an enzyme that breaks the NET backbone can therefore be of use as a therapy for certain conditions.The aim of this thesis was to investigate the mechanisms leading to systemic effects of cancer and potential therapeutic strategies with a special focus on NETs.In paper I we investigated how the presence of a primary tumor affected heart function. We conclude that tumor-induced NETs contribute to inflammation and myocardial strain in malignant disease and identify NETs as potential therapeutic targets in cancer patients to prevent cardiac inflammation and dysfunction.In paper II we investigated if the long-term removal of NETs by the use of a murine DNase I expressing adeno-associated virus vector was possible and how this affected organ function and metastasis formation in tumor-bearing mice. We conclude that long-term, species-specific expression of DNase I is possible. Elevated DNase I expression improved kidney function and reduced the proportion of mice with metastasis.In paper III we investigated how cancer and cancer-associated NETs affect endothelial gene expression in distant organs. We found that the number of differentially expressed genes increased with tumor size and that it was higher in metastatic organs. In response to a tumor, the kidney vasculature showed signs of renal hepatization, a state of kidney stress. In conclusion, this study gives a global overview of the gene expression changes that take place in the vasculature of distant organs in individuals with cancer and the potential role of NETs.
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| 26. |
- Huang, Hua, 1986-, et al.
(författare)
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ELTD1-deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.
- 2021
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Ingår i: Neuro-Oncology. - : Oxford University Press. - 1522-8517 .- 1523-5866. ; 24:3, s. 398-411
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels, and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood.METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1 -/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1-deletion on glioma immunity was determined by treating tumor bearing mice with PD-1-blocking antibodies.RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1-deletion, however, tumor vascular function was improved in ELTD1 -/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1 -/- mice. Consistent with an enhanced inflammatory response, ELTD1-deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade.CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggests that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.
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| 27. |
- Jauhiainen, Suvi, et al.
(författare)
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Proteomics on human cerebral cavernous malformations reveals novel biomarkers in neurovascular dysfunction for the disease pathology
- 2024
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Ingår i: Biochimica et Biophysica Acta - Molecular Basis of Disease. - : Elsevier. - 0925-4439 .- 1879-260X. ; 1870:5
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. Methods: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. Findings: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. Interpretation: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.
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| 28. |
- Johansson, Ulrika, 1974-, et al.
(författare)
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Formation of composite endothelial cell-mesenchymal stem cell islets : a novel approach to promote islet revascularization
- 2008
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 57:9, s. 2393-2401
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Mesenchymal stem cells (MSCs) contribute to endothelial cell (EC) migration by producing proteases, thereby paving the way into the tissues for ECs. MSCs were added to our previously described composite EC islets as a potential means to improve their capacity for islet angiogenesis. RESEARCH DESIGN AND METHODS: Human islets were coated with primary human bone marrow-derived MSCs and dermal microvascular ECs. The capacity of ECs, with or without MSCs, to adhere to and grow into human islets was analyzed. The survival and functionality of these composite islets were evaluated in a dynamic perifusion assay, and their capacity for angiogenesis in vitro was assessed in a three-dimensional fibrin gel assay. RESULTS: ECs proliferated after culture in MSC-conditioned medium, and MSCs improved the EC coverage threefold compared with EC islets alone. Islet survival in vitro and the functionality of the composite islets after culture were equal to those of control islets. The EC-MSC islets showed a twofold increase in total sprout formation compared with EC islets, and vascular sprouts emanating from the EC-MSC-islet surface showed migration of ECs into the islets and also into the surrounding matrix, either alone or in concert with MSCs. CONCLUSIONS: EC proliferation, sprout formation, and ingrowth of ECs into the islets were enhanced by MSCs. The use of composite EC-MSC islets may have beneficial effects on revascularization and immune regulation. The technique presented allows for pretreatment of donor islets with recipient-derived ECs and MSCs as a means of improving islet engraftment.
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| 29. |
- Johansson, Ulrika, 1974-
(författare)
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Formation of Composite Islet Grafts : A novel strategy to promote islet survival and revascularization
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The islets of Langerhans are small and delicate spheroid organs scattered in the pancreas responsible for insulin production. Transplantation of isolated islets is a beneficial therapy for patients with a severe form of type 1 diabetes. The islets, which normally are richly vascularized in the pancreas, are completely disconnected from the vascular support by the enzymatic digestion during the isolation procedure. Islet viability is affected throughout all steps in this process, from donor death and isolation of islets to culturing and the transplantation process itself. In this thesis a novel strategy to promote islet survival and to re-establish islet vasculature is presented. We show endogenous expression of 51 different genes related to inflammation in cultured islets. Among these genes high expression of MCP-1, MIF, VEGF, thymosin b-10 and IL-8, IL-1β, IL-5R-a, IFN-γ antagonist were found in all donors during the 5- and the 2-day cultures, respectively. Protein expression of these genes can stimulate inflammatory immune responses but also promote tissue repair by attracting curative cells such as endothelial cells (EC) leading to re-establishment of the vasculature. We have established a novel technique by formation of composite islets using EC and mesenchymal stem cells (MSC). EC adhered on the surface of the islets and created a potential blood tolerant surface. The EC-islets showed a degree of protection from the detrimental effects of instant blood-mediated inflammatory reaction (IBMIR) with the major components of IBMIR being decreased in in vitro assays. We combined MSC to the EC-islets with success. The MSC were found to have proliferative effect on EC and the combination of these two cell types on the islets further increased the EC covered surface compared to EC-islets. The EC-MSC-islets in co-culture formed vessel-like structures both into the islets and out to the surrounding matrix. The MSC enhanced the exogenous EC to form vessel-like network in the EC-MSC-islets indicating vascular support by the MSC. The novel strategy and conditions presented herein could alleviate problems related to survival of the islets by promoting revascularization. This would open up a new era in islet transplantation and allow more patients to benefit from this therapy.
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| 30. |
- Johansson, Åsa, et al.
(författare)
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Endothelial cell signalling supports pancreatic beta cellfunction in the rat
- 2009
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 52:11, s. 2385-2394
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Tidskriftsartikel (refereegranskat)abstract
- Aims/hypothesis The proximity of endothelial cells andbeta cells in islets by necessity means that they are exposedto each other’s products. Whereas islet endothelial cellsrequire signals from beta cells to function properly,endothelin-1, thrombospondin-1 and laminins, amongothers, have been identified as endothelial-derived molecules,although their full effects on beta cells have not beenexplored. We tested the hypothesis that islet endothelialderivedproducts affect beta cell function.Methods Endothelial cells from rat islets were proliferatedand purified. Endothelium-conditioned culture medium(ECCM) was obtained by maintaining the endothelial cellsin culture medium. Islet function was evaluated followingexposure of cultured islets to standard culture medium orECCM. Changes in mRNA levels for key beta cellmetabolic enzymes were also measured in islets afterECCM exposure.Results Glucose-stimulated insulin release and islet insulincontent were markedly enhanced by exposure to ECCM.This was at least partly explained by improved mitochondrialfunction, as assessed by glucose oxidation and anupregulation of the mitochondrial gene for glycerol-3-phosphate dehydrogenase (mGpdh [also known as Gpd2]),combined with upregulation of the rate-limiting enzyme inthe glycolysis, glucokinase, in the islets. The intracellulardegradation of insulin was also decreased in the islets. Isletendothelial cells produced laminins, and the positive effectsof islet endothelial cells were prevented by addition of aneutralising antibody to the β1-chain of laminin. Additionof exogenous laminin stimulated islet function.Conclusions/interpretation This study provides proof ofprinciple that endothelial cells can affect the function of betacells in their vicinity and that this is at least partially mediatedby laminins.
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| 31. |
- Kourtzelis, Iannis, et al.
(författare)
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Regulation of Instant Blood Mediated Infl ammatoryReaction (IBMIR) in Pancreatic Islet Xeno-Transplantation : Points for Therapeutic Interventions
- 2015
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Ingår i: Immune Responses to Biosurfaces. - Cham : Springer. - 9783319186023 - 9783319186030 ; , s. 171-188
-
Bokkapitel (refereegranskat)abstract
- Xeno-transplantation of pancreatic islets represents a promising therapeuticalternative for the treatment of type 1 diabetes mellitus. However, potentinnate immune responses induced shortly after the transplantation of donor islets tothe recipient, comprising the Instant Blood Mediated Immune Reaction (IBMIR),exert detrimental actions on islet graft function. The coagulation and complementcascades together with the leukocyte and platelet populations are the major playersin IBMIR. This innate immune attack affects dramatically islet integrity and leadsto signifi cant loss of function of the xenograft. In the present review, we focus on themechanisms contributing to IBMIR components and address therapeutic interventionapproaches to limit IBMIR by administering inhibitors in circulation, by coatingthe islet surface with inhibitors or by generating transgenic donor animals; theseapproaches could result in improved xenograft survival.
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| 32. |
- Kourtzelis, Ioannis, et al.
(författare)
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Regulation of Instant Blood Mediated Inflammatory Reaction (IBMIR) in Pancreatic Islet Xeno-Transplantation : Points for Therapeutic Interventions
- 2015
-
Ingår i: IMMUNE RESPONSES TO BIOSURFACES. - Cham : Springer International Publishing. - 9783319186030 - 9783319186023 ; , s. 171-188
-
Konferensbidrag (refereegranskat)abstract
- Xeno-transplantation of pancreatic islets represents a promising therapeutic alternative for the treatment of type 1 diabetes mellitus. However, potent innate immune responses induced shortly after the transplantation of donor islets to the recipient, comprising the Instant Blood Mediated Immune Reaction (IBMIR), exert detrimental actions on islet graft function. The coagulation and complement cascades together with the leukocyte and platelet populations are the major players in IBMIR. This innate immune attack affects dramatically islet integrity and leads to significant loss of function of the xenograft. In the present review, we focus on the mechanisms contributing to IBMIR components and address therapeutic intervention approaches to limit IBMIR by administering inhibitors in circulation, by coating the islet surface with inhibitors or by generating transgenic donor animals; these approaches could result in improved xenograft survival.
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| 33. |
- Lazzaroni, Francesca, et al.
(författare)
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Circulating biomarkers in familial cerebral cavernous malformation
- 2024
-
Ingår i: EBioMedicine. - : Elsevier. - 2352-3964. ; 99
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Tidskriftsartikel (refereegranskat)abstract
- Background Cerebral Cavernous Malformation (CCM) is a rare cerebrovascular disease, characterized by the presence of multiple vascular malformations that may result in intracerebral hemorrhages (ICHs), seizure(s), or focal neurological deficits (FND). Familial CCM (fCCM) is due to loss of function mutations in one of the three independent genes KRIT1 (CCM1), Malcavernin (CCM2), or Programmed Cell death 10 (PDCD10/CCM3). The aim of this study was to identify plasma protein biomarkers of fCCM to assess the severity of the disease and predict its progression.Methods Here, we have investigated plasma samples derived from n = 71 symptomatic fCCM patients (40 female/31 male) and n =17 healthy donors (HD) (9 female/8 male) of the Phase 1/2 Treat_CCM trial, using multiplexed protein profiling approaches.Findings Biomarkers as sCD14 (p = 0.00409), LBP (p = 0.02911), CXCL4 (p = 0.038), ICAM-1 (p = 0.02013), ANG2 (p = 0.026), CCL5 (p = 0.00403), THBS1 (p = 0.0043), CRP (p = 0.0092), and HDL (p = 0.027), were significantly different in fCCM compared to HDs. Of note, sENG (p = 0.011), THBS1 (p = 0.011) and CXCL4 (p = 0.011), were correlated to CCM genotype. sROBO4 (p = 0.014), TM (p = 0.026) and CRP (p = 0.040) were able to predict incident adverse clinical events, such as ICH, FND or seizure. GDF-15, FLT3L, CXCL9, FGF-21 and CDCP1, were identified as predictors of the formation of new MRI-detectable lesions over 2-year follow-up. Furthermore, the functional relevance of ang2, thbs1, robo4 and cdcp1 markers was validated by zebrafish pre-clinical model of fCCM.Interpretation Overall, our study identifies a set of biochemical parameters to predict CCM progression, suggesting biological interpretations and potential therapeutic approaches to CCM disease.
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| 34. |
- Leijon, Jonas, et al.
(författare)
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Attachment of macromolecular heparin conjugate to gelatin scaffolds improves endothelial cell infiltration
- 2013
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Ingår i: Tissue Engineering. Parts A, B and C. - 2152-4947 .- 2152-4955. ; 19:11-12, s. 1336-1348
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Tidskriftsartikel (refereegranskat)abstract
- Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vasculari- zation of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans.
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| 35. |
- Liu, Wei, et al.
(författare)
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Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea : the Connexin26/30 proteins are separately expressed in man
- 2016
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Ingår i: Cell and Tissue Research. - : Springer. - 0302-766X .- 1432-0878. ; , s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This “battery” is located “off-site” in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.
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| 36. |
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| 37. |
- Magnusson, Peetra, et al.
(författare)
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Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1-deficient vascular stem cell development
- 2004
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 117:Pt 8, s. 1513-1523
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Tidskriftsartikel (refereegranskat)abstract
- We have employed embryoid bodies derived from murine embryonal stem cells to study effects on vascular development induced by fibroblast growth factor (FGF)-2 and FGF receptor-1, in comparison to the established angiogenic factor vascular endothelial growth factor (VEGF)-A and its receptor VEGF receptor-2. Exogenous FGF-2 promoted formation of morphologically distinct, long slender vessels in the embryoid bodies, whereas VEGF-A-treated bodies displayed a compact plexus of capillaries. FGF-2 stimulation of embryonal stem cells under conditions where VEGF-A/VEGFR-2 function was blocked, led to formation of endothelial cell clusters, which failed to develop into vessels. FGFR-1(-/-) embryoid bodies responded to VEGF-A by establishment of the characteristic vascular plexus, but FGF-2 had no effect on vascular development in the absence of FGFR-1. The FGFR-1(-/-) embryoid bodies displayed considerably increased basal level of vessel formation, detected by immunohistochemical staining for platelet-endothelial cell adhesion molecule (PECAM)/CD31. This basal vascularization was blocked by neutralizing antibodies against VEGFR-2 or VEGF-A and biochemical analyses indicated changes in regulation of VEGFR-2 in the absence of FGFR-1 expression. We conclude that VEGF-A/VEGFR-2-dependent vessel formation occurs in the absence of FGF-2/FGFR-1, which, however, serve to modulate vascular development.
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| 38. |
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| 39. |
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| 40. |
- Magnusson, Peetra, 1965-
(författare)
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Fibroblast Growth Factor Receptor-1 Function in Vasculo- and Angiogenesis
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During development of the mammalian embryo, spatial and temporal expression of fibroblast growth factors (FGFs) and their cognate receptors are vital in the regulation of a number of patterning processes. Inappropriate or decreased expression leads to severe malformations and even embryonic death. The objectives of this thesis have been to evaluate the usefulness of differentiating embryonic stem (ES) cells as a model to study FGF and FGF receptors in endothelial and hematopoietic cell function in vitro and in vivo, and the effect of an activating mutation in the platelet-derived growth factor receptor-β (PDGFR-β) on endothelial cells and vessel formation.Aggregates of differentiating ES cells, denoted embryoid bodies, faithfully recapitulate many developmental processes. Embryoid bodies cultured in fetal calf serum spontaneously develop cardiomyocytes and endothelial cells. The endothelial cells organize into lumen-containing vessels carrying erythroblasts. Administration of FGF or vascular endothelial growth factor (VEGF)-A promotes development of specific vascular phenotypes. About 20% of endothelial cells in embryoid bodies and teratomas express FGFR-1, and these FGFR-1-expressing endothelial cells are mitogenically active in the absence of exogenous stimuli and respond to VEGF-A to the same extent as endothelial cells lacking FGFR-1 expression. FGFR-1 deficiency leads to arrest in hematopoietic differentiation, whereas endothelial cell development is enhanced. As a consequence, teratomas derived from ES cells lacking FGFR-1 expression display vessels composed of a double layer of endothelial cells. The hyperactivity of endothelial cells derived from FGFR-1-deficient ES cells is suggested to be due to hyperactivity of VEGF receptor-2, as well as to loss of negative regulators of angiogenesis, such as interleukin-4.Mutation of platelet-derived factor receptor-β (PDGFR-β) to replace D849 in the activating loop in the kinase domain with V leads to ligand-independent kinase activity, increased basal signal transduction, and enhanced expression of VEGF-A as well as VEGFR-2. As a result, endothelial cell sprouts covered with pericyte-like cells are formed in a VEGF-A/VEGFR-2 dependent manner in ES cells expressing the mutated PDGFR-β.In conclusion, embryoid bodies represent a high-quality model for the study of growth factor-regulated vascular development and sprouting angiogenesis.
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| 41. |
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| 42. |
- Magnusson, Peetra U, et al.
(författare)
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FGFR-1 regulates angiogenesis through cytokines interleukin-4 and pleiotrophin
- 2007
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 110:13, s. 4214-4222
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Tidskriftsartikel (refereegranskat)abstract
- The role of fibroblast growth factors (FGFs) in blood vessel formation has remained unclear. We used differentiating stem-cell cultures (embryoid bodies) and teratomas to show that FGIF receptor-1 (FGFR-1) exerts a negative regulatory effect on endothelial cell function in these models. Embryoid bodies lacking expression of FGFR-1 as a result of gene targeting (Fgfr-1(-/-)) displayed increased vascularization and a distinct, elongated vessel morphology. Teratomas derived from FGFR-1-deficient stem cells were characterized by an increased growth rate and abundant, morphologically distinct vessels. Transmission electron microscopy of the Fgfr-1(-/-) teratomas showed a compact and voluminous but functional endothelium, which anastomosed with the host circulation. The increased vascularization and altered endothelial cell morphology was dependent on secreted factor(s), based on the transfer of the Fgfr-1(+/-) vascular phenotype by conditioned medium to Fgfr-1(+/-) embryoid bodies. Antibody and transcript arrays showed down-regulation of interleukin-4 (IL-4) and up-regulation of pleiotrophin in Fgfr-1(-/-) embryoid bodies, compared with the heterozygous cultures. We used neutralizing antibodies to show that IL-4 and pleiotrophin act as negative and positive angiogenic regulators, respectively. We conclude that FGFR-1 negatively regulates endothelial cell function by altering the balance of modulatory cytokines.
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| 43. |
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| 44. |
- Magnusson, Peetra U., et al.
(författare)
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Platelet-derived growth factor receptor-beta constitutive activity promotes angiogenesis in vivo and in vitro
- 2007
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 27:10, s. 2142-2149
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE - Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-β (PDGFR-β), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-β (Pdgfrb) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo. METHODS AND RESULTS - Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-β tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. Pdgfrb embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, Pdgfrb embryonic stem cell-derived teratomas in nude mice were more densely vascularized than wild-type teratomas. CONCLUSION - Increased PDGFR-β kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.
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| 45. |
- Malinverno, Matteo, et al.
(författare)
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Endothelial cell clonal expansion in the development of cerebral cavernous malformations
- 2019
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics.
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| 46. |
- Matsumoto, Taro, et al.
(författare)
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VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis.
- 2005
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Ingår i: EMBO J. - 0261-4189. ; 24:13, s. 2342-53
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis.
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| 47. |
- Moll, Guido, et al.
(författare)
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Are Therapeutic Human Mesenchymal Stromal Cells Compatible with Human Blood?
- 2012
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 30:7, s. 1565-1574
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Tidskriftsartikel (refereegranskat)abstract
- Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture-expanded human MSCs elicit an innate immune attack, termed instant blood-mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell-passage number. Short-term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D-dimer or acute-phase reactants with the currently applied dose of 1.0-3.0 x 10(6) cells per kilogram. Culture-expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose-dependent and increases after prolonged ex vivo expansion. Currently applied doses of low-passage clinical-grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells.
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| 48. |
- Nilsson Ekdahl, Kristina, et al.
(författare)
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Dangerous liaisons : complement, coagulation, and kallikrein/kinin cross-talk act as a linchpin in the events leading to thromboinflammation
- 2016
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Ingår i: Immunological Reviews. - : Wiley. - 0105-2896 .- 1600-065X. ; 274:1, s. 245-269
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Forskningsöversikt (refereegranskat)abstract
- Innate immunity is fundamental to our defense against microorganisms. Physiologically, the intravascular innate immune system acts as a purging system that identifies and removes foreign substances leading to thromboinflammatory responses, tissue remodeling, and repair. It is also a key contributor to the adverse effects observed in many diseases and therapies involving biomaterials and therapeutic cells/organs. The intravascular innate immune system consists of the cascade systems of the blood (the complement, contact, coagulation, and fibrinolytic systems), the blood cells (polymorphonuclear cells, monocytes, platelets), and the endothelial cell lining of the vessels. Activation of the intravascular innate immune system in vivo leads to thromboinflammation that can be activated by several of the system's pathways and that initiates repair after tissue damage and leads to adverse reactions in several disorders and treatment modalities. In this review, we summarize the current knowledge in the field and discuss the obstacles that exist in order to study the cross-talk between the components of the intravascular innate immune system. These include the use of purified in vitro systems, animal models and various types of anticoagulants. In order to avoid some of these obstacles we have developed specialized human whole blood models that allow investigation of the cross-talk between the various cascade systems and the blood cells. We in particular stress that platelets are involved in these interactions and that the lectin pathway of the complement system is an emerging part of innate immunity that interacts with the contact/coagulation system. Understanding the resulting thromboinflammation will allow development of new therapeutic modalities.
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| 49. |
- Nilsson Ekdahl, Kristina, et al.
(författare)
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Thromboinflammation in Therapeutic Medicine
- 2015
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Ingår i: Advances in Experimental Medicine and Biology. - Cham : Springer International Publishing. - 0065-2598 .- 2214-8019. - 9783319186023 - 9783319186030 ; 865, s. 3-17
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Forskningsöversikt (refereegranskat)abstract
- Thromboinflammation is primarily triggered by the humoral innate immune system, which mainly consists of the cascade systems of the blood, i.e., the complement, contact/coagulation and fibrinolytic systems. Activation of these systems subsequently induces activation of endothelial cells, leukocytes and platelets, finally resulting in thrombotic and inflammatory reactions. Such reactions are triggered by a number of medical procedures, e.g., treatment with biomaterials or drug delivery devices as well as in transplantation with cells, cell clusters or whole vascularized organs. Here, we (1) describe basic mechanisms for thromboinflammation; (2) review thromboinflammatory reactions in therapeutic medicine; and (3) discuss emerging strategies to dampen thromboinflammation.
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| 50. |
- Nilsson, Per H., et al.
(författare)
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Autoregulation of thromboinflammation on biomaterial surfaces by a multicomponent therapeutic coating
- 2013
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Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 34:4, s. 985-994
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Tidskriftsartikel (refereegranskat)abstract
- Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H-binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation.
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