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| 2. |
- Björquist, P, et al.
(author)
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Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator.
- 1994
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In: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1209:2, s. 191-202
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Journal article (peer-reviewed)abstract
- Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)
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| 3. |
- Brohlin, Maria, 1966-, et al.
(author)
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Effects of a defined xeno-free medium on the growth and neurotrophic and angiogenic properties of human adult stem cells
- 2017
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In: Cytotherapy. - : ELSEVIER SCI LTD. - 1465-3249 .- 1477-2566. ; 19:5, s. 629-639
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Journal article (peer-reviewed)abstract
- Background. The growth properties and neurotrophic and angiogenic effects of human mesenchymal stromal cells (MSCs) cultured in a defined xeno-free, serum-free medium (MesenCult-XF) were investigated. Methods. Human MSCs from adipose tissue (ASCs) and bone marrow (BMSCs) were cultured in Minimum Essential Medium-alpha (alpha-MEM) containing fetal calf serum or in MesenCult-XF. Proliferation was measured over 10 passages and the colony-forming unit (CFU) assay and expression of cluster of differentiation (CD) surface markers were determined. Neurite outgrowth and angiogenic activity of the MSCs were determined. Results. At early passage, both ASCs and BMSCs showed better proliferation in MesenCult-XF compared with standard a-MEM containing serum. However, CFUs were significantly lower in MesenCult-XF. ASCs cultured in MesenCult-XF continued to expand at faster rates than cells grown in serum. BMSCs showed morphological changes at late passage in MesenCult-XF and stained positive for senescence beta-galactosidase activity. Expression levels of CD73 and CD90 were similar in both cell types under the various culture conditions but CD105 was significantly reduced at passage 10 in MesenCult-XF. In vitro stimulation of the cells enhanced the expression of brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF-A) and angiopoietin-1. Stimulated ASCs grown in MesenCult-XF evoked the longest neurite outgrowth in a neuron co-culture model. Stimulated BMSCs grown in MesenCult-XF produced the most extensive network of capillary-like tube structures in an in vitro angiogenesis assay. Conclusions. ASCs and BMSCs exhibit high levels of neurotrophic and angiogenic activity when grown in the defined serum free medium indicating their suitability for treatment of various neurological conditions. However, long-term expansion in MesenCult-XF might be restricted to ASCs.
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| 4. |
- Cheng, X F, et al.
(author)
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Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA.
- 1995
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In: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 77:2, s. 149-64
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Journal article (peer-reviewed)abstract
- The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.
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| 5. |
- Kumar Kuna, Vijay, et al.
(author)
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Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization
- 2022
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:15
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Journal article (peer-reviewed)abstract
- Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.
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| 6. |
- Lauvrud, Anne Therese, 1975-
(author)
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Optimizing stem cells for reconstructive surgery
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Fat grafting has become an established method in plastic surgery for treating soft tissue defects. The results for survival of the fat being transplanted is unpredictable and supplementation of the graft with the Stromal Vascular Fraction (SVF) or cultures Adipose tissue-derived stem cells (ASCs) can enhance graft viability. The ASCs are a heterogenous group of cells with various cell membrane markers, and differing growth promoting and differentiation characteristics of the stem cells derived from the fat. It is of high importance when expanding cells prior to the transplantation of the cells into patients, that the culture conditions are well defined and ideally are xenofree, avoiding use of animal-derived products. Furthermore, the procedures must be safe and not increase risk for recurrence of cancer after reconstructive surgeries. This thesis explores the phenotypic properties of a selected population of ASCs, with a view to determining their suitability for transplantation into fat grafts. ASCs were isolated from SVF of human abdominal fat and CD146+ cells were selected using immunomagnetic beads. The proliferation, angiogenic and adipogenic properties were significantly higher in the CD146+ cells. Stem cells were also isolated from lipoaspirate obtained using two different liposuction methods. Waterjet lipoaspirates yielded the greatest number of CD146+ cells with high adipogenic potential and angiogenic activity. The cells could also be successfully isolated using a closed processing system. Cells were expanded in either foetal bovine serum, platelet lysate or a chemically defined xenofree (XV) medium. Cultures in XV medium proliferated the fastest, expressed the highest number of CD146+ cells, and showed the best adipogenic and angiogenic properties. To test possible ASCs interactions with cancer cells, co-cultures with MCF-7 breast cancer cells were established. Conditioned medium from co-cultures significantly increased the migration of the cancer cells but not their proliferation, and there was increased expression of Tenascin-C in these cultures. The research in this thesis work has shown more optimal ways to isolate and expand ASCs, potentially offering new therapeutic reconstructive treatment options for a variety of medical conditions.
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| 7. |
- Lauvrud, Anne Therese, et al.
(author)
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Water jet-assisted lipoaspiration and Sepax cell separation system for the isolation of adipose stem cells with high adipogenic potential
- 2021
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In: Journal of Plastic, Reconstructive & Aesthetic Surgery. - : Elsevier. - 1748-6815 .- 1878-0539. ; 74:10, s. 2759-2767
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Journal article (peer-reviewed)abstract
- Introduction: Water jet-assisted liposuction has gained popularity due to favourable fat grafting outcomes. In this study, we compared stem cells obtained from fat isolated with manual or the water jet-assisted procedure.Methods: Liposuction of abdominal fat was performed using the two methods on each donor (n = 10). Aspirate samples were collagenase digested and the isolated cells seeded in vitro prior to proliferation, adipogenic differentiation and angiogenic activity analyses.Results: Cells from either procedure proliferated at similar rates and exhibited a similar colony-forming ability. The cells expressed stem cell markers CD73, CD90 and CD105. In the water jet cell preparations, there were higher numbers of cells expressing CD146. Robust adipogenic differentiation was observed in cultures expanded from both manual and water jet lipoaspirates. Gene analysis showed higher expression of the adipocyte markers aP2 and GLUT4 in the adipocyte-differentiated water jet cell preparations, and ELISA indicated increased secretion of adiponectin from these cells. Both cell groups expressed vasculogenic factors and the water jet cells promoted the highest levels of in vitro angiogenesis. Given these positive results, we further characterised the water jet cells when prepared using an automated closed cell processing unit, the Sepax-2 system (Cytiva). The growth and stem cell properties of the Sepax-processed cells were similar to the standard centrifugation protocol, but there was evidence for greater adipogenic differentiation in the Sepax-processed cells.Conclusions: Water jet lipoaspirates yield cells with high adipogenic potential and angiogenic activity, which may be beneficial for use in cell-assisted lipotransfers.
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| 8. |
- Qu, Chengjuan, 1967-, et al.
(author)
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Evaluation of growth, stemness, and angiogenic properties of dental pulp stem cells cultured in cGMP xeno-/serum-free medium
- 2020
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In: Cell and Tissue Research. - : Springer. - 0302-766X .- 1432-0878. ; 380, s. 93-105
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Journal article (peer-reviewed)abstract
- This study was aimed to investigate the effects of cGMP xeno-/serum-free medium (XSF, Irvine Scientific) on the properties of human dental pulp stem cells (DPSCs). DPSCs, from passage 2, were cultured in XSF or fetal bovine serum (FBS)-supplemented medium, and sub-cultured up to passage 8. Cumulative population doublings (PDs) and the number of colony-forming-units (CFUs) were determined. qRT-PCR, ELISA, and in vitro assays were used to assess angiogenic capacity. Flow cytometry was used to measure CD73, CD90, and CD105 expression. Differentiation into osteo-, adipo-, and chondrogenic cell lineages was performed. DPSCs showed more elongated morphology, a reduced rate of proliferation at later passages, and lower CFU counts in XSF compared with FBS. Expression of angiogenic factors at the gene and protein levels varied in the two media and with passage number, but cells grown in XSF had more in vitro angiogenic activity. The majority of early and late passage DPSCs cultured in XSF expressed CD73 and CD90. In contrast, the percentage of CD105 positive DPSCs in XSF medium was significantly lower with increased passage whereas the majority of cells cultured in FBS were CD105 positive. Switching XSF-cultured DPSCs to medium supplemented with human serum restored the expression of CD105. The tri-lineage differentiation of DPSCs cultured under XSF and FBS conditions was similar. We showed that despite reduced CD105 expression levels, DPSCs expanded in XSF medium maintained a functional MSC phenotype. Furthermore, restoration of CD105 expression is likely to occur upon in vivo transplantation, when cells are exposed to human serum.
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