| 1. |
- Bergwik, Jesper, et al.
(författare)
-
A novel quinoline with airway relaxant effects and anti-inflammatory properties
- 2024
-
Ingår i: Respiratory Research. - 1465-9921 .- 1465-993X. ; 25:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: In chronic pulmonary diseases characterized by inflammation and airway obstruction, such as asthma and COPD, there are unmet needs for improved treatment. Quinolines is a group of small heterocyclic compounds that have a broad range of pharmacological properties. Here, we investigated the airway relaxant and anti-inflammatory properties of a novel quinoline (RCD405). Methods: The airway relaxant effect of RCD405 was examined in isolated airways from humans, dogs, rats and mice. Murine models of ovalbumin (OVA)-induced allergic asthma and LPS-induced airway inflammation were used to study the effects in vivo. RCD405 (10 mg/kg) or, for comparisons in selected studies, budesonide (3 mg/kg), were administered intratracheally 1 h prior to each challenge. Airway responsiveness was determined using methacholine provocation. Immune cell recruitment to bronchi was measured using flow cytometry and histological analyses were applied to investigate cell influx and goblet cell hyperplasia of the airways. Furthermore, production of cytokines and chemokines was measured using a multiplex immunoassay. The expression levels of asthma-related genes in murine lung tissue were determined by PCR. The involvement of NF-κB and metabolic activity was measured in the human monocytic cell line THP-1. Results: RCD405 demonstrated a relaxant effect on carbachol precontracted airways in all four species investigated (potency ranking: human = rat > dog = mouse). The OVA-specific IgE and airway hyperresponsiveness (AHR) were significantly reduced by intratracheal treatment with RCD405, while no significant changes were observed for budesonide. In addition, administration of RCD405 to mice significantly decreased the expression of proinflammatory cytokines and chemokines as well as recruitment of immune cells to the lungs in both OVA- and LPS-induced airway inflammation, with a similar effect as for budesonide (in the OVA-model). However, the effect on gene expression of Il-4, IL-5 and Il-13 was more pronounced for RCD405 as compared to budesonide. Finally, in vitro, RCD405 reduced the LPS-induced NF-κB activation and by itself reduced cellular metabolism. Conclusions: RCD405 has airway relaxant effects, and it reduces AHR as well as airway inflammation in the models used, suggesting that it could be a clinically relevant compound to treat inflammatory airway diseases. Possible targets of this compound are complexes of mitochondrial oxidative phosphorylation, resulting in decreased metabolic activity of targeted cells as well as through pathways associated to NF-κB. However, further studies are needed to elucidate the mode of action.
|
|
| 2. |
- Bergwik, Jesper, et al.
(författare)
-
Binding of the human antioxidation protein α1-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro
- 2021
-
Ingår i: Redox Biology. - : Elsevier BV. - 2213-2317. ; 41
-
Tidskriftsartikel (refereegranskat)abstract
- Heparin and heparan sulfate (HS) are linear sulfated disaccharide polymers. Heparin is found mainly in mast cells, while heparan sulfate is found in connective tissue, extracellular matrix and on cell membranes in most tissues. α1-microglobulin (A1M) is a ubiquitous protein with thiol-dependent antioxidant properties, protecting cells and matrix against oxidative damage due to its reductase activities and radical- and heme-binding properties. In this work, it was shown that A1M binds to heparin and HS and can be purified from human plasma by heparin affinity chromatography and size exclusion chromatography. The binding strength is inversely dependent of salt concentration and proportional to the degree of sulfation of heparin and HS. Potential heparin binding sites, located on the outside of the barrel-shaped A1M molecule, were determined using hydrogen deuterium exchange mass spectrometry (HDX-MS). Immunostaining of endothelial cells revealed pericellular co-localization of A1M and HS and the staining of A1M was almost completely abolished after treatment with heparinase. A1M and HS were also found to be co-localized in vivo in the lungs, aorta, kidneys and skin of mice. The redox-active thiol group of A1M was unaffected by the binding to HS, and the cell protection and heme-binding abilities of A1M were slightly affected. The discovery of the binding of A1M to heparin and HS provides new insights into the biological role of A1M and represents the basis for a novel method for purification of A1M from plasma.
|
|
| 3. |
- Bergwik, Jesper, et al.
(författare)
-
Knockout of the radical scavenger α1-microglobulin in mice results in defective bikunin synthesis, endoplasmic reticulum stress and increased body weight
- 2021
-
Ingår i: Free Radical Biology and Medicine. - : Elsevier BV. - 0891-5849. ; 162
-
Tidskriftsartikel (refereegranskat)abstract
- α1-microglobulin (A1M) is a ubiquitous protein with reductase and radical- and heme-binding properties. The protein is mainly expressed in the liver and encoded by the α1-microglobulin-bikunin precursor (AMBP) gene together with the plasma proteinase inhibitor bikunin. The AMBP polypeptide is translated, glycosylated and the C-terminal bikunin part linked via a chondroitin sulfate glycosaminoglycan chain to one or two heavy chains in the endoplasmic reticulum (ER) and Golgi compartments. After proteolytic cleavage, the A1M protein and complexed bikunin parts are secreted separately. The complete physiological role of A1M, and the reason for the co-synthesis with bikunin, are both still unknown. The aim of this work was to develop an A1M knockout (A1M−KO) mouse model lacking expression of A1M, but with a preserved bikunin expression, and to study the phenotypic traits in these mice, with a focus on hepatic endoplasmic reticulum (ER) function. The bikunin expression was increased in the A1M−KO mouse livers, while the bikunin levels in plasma were decreased, indicating a defective biosynthesis of bikunin. The A1M−KO livers also showed an increased expression of transducers of the unfolded protein response (UPR), indicating an increased ER-stress in the livers. At twelve months of age, the A1M−KO mice also displayed an increased body weight, and an increased liver weight and lipid accumulation. Moreover, the KO mice showed an increased expression of endogenous antioxidants in the liver, but not in the kidneys. Together, these results suggest a physiological role of A1M as a regulator of the intracellular redox environment and more specifically the ER folding and posttranslational modification processes, particularly in the liver.
|
|
| 4. |
- Bergwik, Jesper, et al.
(författare)
-
Macrophage expressed tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis progression
- 2024
-
Ingår i: Immunology. - 0019-2805. ; 171:4, s. 583-594
-
Tidskriftsartikel (refereegranskat)abstract
- Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder involving scarring of pulmonary tissue and a subsequent decrease in respiratory capacity, ultimately resulting in death. Tartrate resistant acid phosphatase 5 (ACP5) plays a role in IPF but the exact mechanisms are yet to be elucidated. In this study, we have utilized various perturbations of the bleomycin mouse model of IPF including genetic knockout, RANKL inhibition, and macrophage adoptive transfer to further understand ACP5's role in pulmonary fibrosis. Genetic ablation of Acp5 decreased immune cell recruitment to the lungs and reduced the levels of hydroxyproline (reflecting extracellular matrix-production) as well as histological damage. Additionally, gene expression profiling of murine lung tissue revealed downregulation of genes including Ccl13, Mmp13, and Il-1α that encodes proteins specifically related to immune cell recruitment and macrophage/fibroblast interactions. Furthermore, antibody-based neutralization of RANKL, an important inducer of Acp5 expression, reduced immune cell recruitment but did not decrease fibrotic lung development. Adoptive transfer of Acp5 -/- bone marrow-derived monocyte (BMDM) macrophages 7 or 14 days after bleomycin administration resulted in reductions of cytokine production and decreased levels of lung damage, compared to adoptive transfer of WT control macrophages. Taken together, the data presented in this study suggest that macrophage derived ACP5 plays an important role in development of pulmonary fibrosis and could present a tractable target for therapeutic intervention in IPF.
|
|
| 5. |
- Bergwik, Jesper, et al.
(författare)
-
Structure, Functions, and Physiological Roles of the Lipocalin α1-Microglobulin (A1M)
- 2021
-
Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 12
-
Forskningsöversikt (refereegranskat)abstract
- α1-microglobulin (A1M) is found in all vertebrates including humans. A1M was, together with retinol-binding protein and β-lactoglobulin, one of the three original lipocalins when the family first was proposed in 1985. A1M is described as an antioxidant and tissue cleaning protein with reductase, heme- and radical-binding activities. These biochemical properties are driven by a strongly electronegative surface-exposed thiol group, C34, on loop 1 of the open end of the lipocalin barrel. A1M has been shown to have protective effects in vitro and in vivo in cell-, organ-, and animal models of oxidative stress-related medical conditions. The gene coding for A1M is unique among lipocalins since it is flanked downstream by four exons coding for another non-lipocalin protein, bikunin, and is consequently named α1-microglobulin-bikunin precursor gene (AMBP). The precursor is cleaved in the Golgi, and A1M and bikunin are secreted from the cell separately. Recent publications have suggested novel physiological roles of A1M in regulation of endoplasmic reticulum activities and erythrocyte homeostasis. This review summarizes the present knowledge of the structure and functions of the lipocalin A1M and presents a current model of its biological role(s).
|
|
| 6. |
|
|
| 7. |
- Bergwik, Jesper, et al.
(författare)
-
α1-Microglobulin Binds Illuminated Flavins and Has a Protective Effect Against Sublethal Riboflavin-Induced Damage in Retinal Epithelial Cells
- 2020
-
Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- Riboflavin (vitamin B2) is an important constituent of the prosthetic groups flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are utilized as electron-carriers in energy metabolism. Excitation by UV-light leads to the generation of riboflavin radicals and reactive oxygen species (ROS), which can oxidize a wide range of biomolecules. The human protein α1-microglobulin (A1M) is a reductase and a radical scavenger, which can protect cells and matrix against oxidative damage. Here, we provide evidence of a molecular interaction between illuminated riboflavin and A1M, similar to the radical scavenging reactions previously seen between A1M and other organic radicals. Binding between riboflavin and A1M was demonstrated by gel migration shift, UV-absorbance and fluorescence spectrum analysis. The reaction between A1M and UV-light illuminated riboflavin involved covalent modification of A1M and proteolytic release of an N-terminal part of the protein. Furthermore, A1M also inhibited the ROS-induced photoreduction reaction of riboflavin, in a reaction involving the free thiol group in position C34. Finally, the results show a protective effect of A1M, analyzed by gene expression rates of stress genes, against sublethal damage in retinal epithelial cells in culture. Together, our results suggest a new role of A1M as a scavenger of riboflavin radicals and ROS produced during illumination of riboflavin.
|
|
| 8. |
- Carlsson, Magnus L.R., et al.
(författare)
-
Expression, Purification and Initial Characterization of Functional α1-Microglobulin (A1M) in Nicotiana benthamiana
- 2020
-
Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.
|
|
| 9. |
- Ghosh, Fredrik, et al.
(författare)
-
Acute tissue reactions, inner segment pathology, and effects of the antioxidant α1-microglobulin in an in vitro model of retinal detachment
- 2018
-
Ingår i: Experimental Eye Research. - : Elsevier BV. - 0014-4835. ; 173, s. 13-23
-
Tidskriftsartikel (refereegranskat)abstract
- The purpose of this study was to explore acute tissue reactions, ultrastructural photoreceptor morphology with emphasis on inner segments, and the effect of antioxidant treatment in an in vitro model of rhegmatogenous retinal detachment (RRD). A previously described method of RRD simulation was used with adult retinal porcine explants kept free-floating in culture medium with or without treatment with the radical scavenger α1-microglobulin (A1M). Explants were examined at 5 time points from 1 to 24 h using transmission electron microscopy as well as quantitative real-time PCR (RT-PCR) to quantify gene expression of the cell stress marker heat shock protein 70 (Hsp70) and oxidative stress marker heme oxygenase (HO-1). The culture medium level of the cell damage marker lactate dehydrogenase (LDH) and oxidative stress DNA damage marker 8-Oxo-2'-deoxyguanosine (8-OHdG) was also assessed at each time point. We found that the levels of Hsp70 and LDH rapidly increased in both groups, and at 3 and 6 h, Hsp70 was significantly higher in A1M treated retinas. At 24 h, Hsp70 and LDH, as well as 8-OHdG were significantly lower compared with controls, whereas the tissue level of HO-1 was significantly higher. Progressive ultrastructural photoreceptor changes were seen in untreated control explants from 1 h and onwards including outer segment shortening and loss, disruption of organelles within the inner segments and loss of perikarya in the outer nuclear layer. Inner segment pathology was more rapid and extensive in rods compared with in cones. In A1M treated counterparts, damage to rod inner segment mitochondria was significantly higher after 1 h of culture, but after this time, no statistical difference was found. At 24 h, cone inner segment mitochondrial disruption was significantly higher in control retinas and the number of surviving perikarya lower. From our results, we conclude that retinal explants elicit acute cell stress reactions when placed in culture without physical support simulating a detached retina floating in the vitreous space. Photoreceptors rapidly display degenerative changes including extensive damage to inner segment mitochondria indicating loss of energy transduction as an early key event. A1M increases initial mitochondrial stress in the rods, however, subsequent pathology is attenuated by the treatment, highlighting the dynamics of protective as well as disruptive oxidative stress reactions in the detached retina.
|
|
| 10. |
- Kristiansson, Amanda, et al.
(författare)
-
Human radical scavenger α1-microglobulin protects against hemolysis in vitro and α1-microglobulin knockout mice exhibit a macrocytic anemia phenotype
- 2021
-
Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849. ; 162
-
Tidskriftsartikel (refereegranskat)abstract
- During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α1-microglobulin (A1M) is a ubiquitous reductase and radical- and heme-binding protein with antioxidation properties. Although present in the circulation in micromolar concentrations, its function in blood is unclear. Here, we show that A1M provides RBC stability. A1M-/- mice display abnormal RBC morphology, reminiscent of macrocytic anemia conditions, i.e. fewer, larger and more heterogeneous cells. Recombinant human A1M (rA1M) reduced in vitro hemolysis of murine RBC against spontaneous, osmotic and heme-induced stress. Moreover, A1M is taken up by human RBCs both in vitro and in vivo. Similarly, rA1M also protected human RBCs against in vitro spontaneous, osmotic, heme- and radical-induced hemolysis as shown by significantly reduced leakage of hemoglobin and LDH. Addition of rA1M resulted in decreased hemolysis compared to addition of the heme-binding protein hemopexin and the radical-scavenging and reducing agents ascorbic acid and Trolox (vitamin E). Furthermore, rA1M significantly reduced spontaneous and heme-induced fetal RBC cell death. Addition of A1M to human whole blood resulted in a significant reduction of hemolysis, whereas removal of A1M from whole blood resulted in increased hemolysis. We conclude that A1M has a protective function in reducing hemolysis which is neither specific to the origin of hemolytic insult, nor species specific.
|
|
