| 1. |
- Davidsson, Pia, et al.
(author)
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Vascular endothelial growth factor-D plasma levels and VEGFD genetic variants are independently associated with outcomes in patients with cardiovascular disease
- 2023
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In: Cardiovascular Research. - : Oxford University Press. - 0008-6363 .- 1755-3245. ; 119:7, s. 1596-1605
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Journal article (peer-reviewed)abstract
- Aims The vascular endothelial growth factor (VEGF) family is involved in pathophysiological mechanisms underlying cardiovascular (CV) diseases. The aim of this study was to investigate the associations between circulating VEGF ligands and/or soluble receptors and CV outcome in patients with acute coronary syndrome (ACS) and chronic coronary syndrome (CCS).Methods and results Levels of VEGF biomarkers, including bFGF, Flt-1, KDR (VEGFR2), PlGF, Tie-2, VEGF-A, VEGF-C, and VEGF-D, were measured in the PLATO ACS cohort (n = 2091, discovery cohort). Subsequently, VEGF-D was also measured in the STABILITY CCS cohort (n = 4015, confirmation cohort) to verify associations with CV outcomes. Associations between plasma VEGF-D and outcomes were analysed by multiple Cox regression models with hazard ratios (HR [95% CI]) comparing the upper vs. the lower quartile of VEGF-D. Genome-wide association study (GWAS) of VEGF-D in PLATO identified SNPs that were used as genetic instruments in Mendelian randomization (MR) meta-analyses vs. clinical endpoints. GWAS and MR were performed in patients with ACS from PLATO (n = 10 013) and FRISC-II (n = 2952), and with CCS from the STABILITY trial (n = 10 786). VEGF-D, KDR, Flt-1, and PlGF showed significant association with CV outcomes. VEGF-D was most strongly associated with CV death (P = 3.73e-05, HR 1.892 [1.419, 2.522]). Genome-wide significant associations with VEGF-D levels were identified at the VEGFD locus on chromosome Xp22. MR analyses of the combined top ranked SNPs (GWAS P-values; rs192812042, P = 5.82e-20; rs234500, P = 1.97e-14) demonstrated a significant effect on CV mortality [P = 0.0257, HR 1.81 (1.07, 3.04) per increase of one unit in log VEGF-D].Conclusion This is the first large-scale cohort study to demonstrate that both VEGF-D plasma levels and VEGFD genetic variants are independently associated with CV outcomes in patients with ACS and CCS. Measurements of VEGF-D levels and/or VEGFD genetic variants may provide incremental prognostic information in patients with ACS and CCS.
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| 2. |
- Bost, Jeremy P., et al.
(author)
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Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
- 2022
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
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Journal article (peer-reviewed)abstract
- The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
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| 3. |
- Collén, Anna, et al.
(author)
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A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I.
- 2002
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In: Biochimica et Biophysica Acta. - 0006-3002. ; 1569:1-3, s. 139-150
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Journal article (peer-reviewed)abstract
- Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.
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| 4. |
- Collen, Anna-Clara, 1970, et al.
(author)
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Cardiac structure and function is related to current blood pressure rather than to previous hypertensive pregnancy
- 2015
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In: Journal of Human Hypertension. - : Springer Science and Business Media LLC. - 0950-9240 .- 1476-5527. ; 29:11, s. 702-704
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Journal article (peer-reviewed)abstract
- One-hundred five women were examined with echocardiography and ambulatory blood pressure measurements 40 years post partum to evaluate the effect of former hypertensive pregnancies versus current blood pressure on cardiac structure and function. Hypertensive pregnancies did not have an adverse effect on the heart, but current minor elevation in blood pressure had a negative impact on the myocardium. The increased prevalence of hypertension following hypertensive pregnancies may be a crucial factor regarding the increased risk for cardiovascular disease shown in affected women.
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| 5. |
- Collen, Anna-Clara, 1970, et al.
(author)
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Cardiovascular and metabolic characteristics 40 years after hypertensive pregnancies: a long-term follow-up study of mothers.
- 2013
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In: Journal of hypertension. - 1473-5598. ; 31:4, s. 758-765
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Journal article (peer-reviewed)abstract
- OBJECTIVES:: Maternal cardiovascular morbidity is increased after hypertensive pregnancies (HTP). The pathways from complicated pregnancies to future cardiovascular disease are complex. The aim of the present study was to test the hypothesis that different cardiovascular mechanisms are changed in women who experienced HTP four decades earlier in comparison to women with normotensive pregnancies. METHODS:: One hundred and five women (50 with hypertensive and 55 with normal pregnancies) were examined with anthropometric measurements; office blood pressure, ambulatory blood pressure and central blood pressure, pulse wave velocity, augmentation index, intimal-media thickness, echocardiography and laboratory measurements. In addition another 204 women were followed-up by a questionnaire regarding their pregnancy 40 years ago, as well as their present health status and medications. RESULTS:: Women with HTP had more often diagnosed hypertension when compared with women with normal pregnancies (50 vs. 31%, respectively; P=0.046), but the groups did not differ in any blood pressure levels. HTP were associated with higher pulse wave velocity (8.8m/s vs. 7.8m/s, P=0.021), and higher levels of P-glucose (5.7mmol/l vs. 5.2mmol/l, P=0.022), P-HbA1c (4.4% vs. 4.2%, P=0.010) and noradrenaline (2.45mmol/l vs. 2.11mmol/l, P=0.040) when compared with normotensive pregnancies. Women followed up with a questionnaire reported deteriorated cardiovascular health compared to women attending the clinical investigations of the study. CONCLUSION:: HTP are associated with impairment in vascular function and metabolic status 40 years postpartum despite well controlled blood pressure levels.
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| 6. |
- Collen, Anna-Clara, 1970, et al.
(author)
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Cardiovascular response to stress and perceived stress is not altered 40 years after hypertensive pregnancies
- 2015
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In: Hypertension in Pregnancy. - : Informa UK Limited. - 1064-1955 .- 1525-6065. ; 34:1, s. 116-124
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Journal article (peer-reviewed)abstract
- Objective: Women experiencing hypertensive pregnancies have an increased risk for cardiovascular disease. Whether stress increase the risk is unknown. The objective was to test if cardiovascular response to stress and/or perceived stress differed in relation to blood pressure status during pregnancy 40 years earlier. Methods: Cardiovascular response was examined with mental stress test, and perceived stress was evaluated with a questionnaire in 105 women. Results: Resting heart rate was higher, and pulse reactivity was lower in women with previous hypertensive pregnancies. Neither blood pressure nor perceived stress differed. Conclusion: Response to physical or psychological stress is not affected many years after pregnancy.
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| 7. |
- Collen, Anna-Clara, 1970, et al.
(author)
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Sympathetic nerve activity in women 40 years after a hypertensive pregnancy
- 2012
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In: Journal of hypertension. - 1473-5598. ; 30:6, s. 1203-1210
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Journal article (peer-reviewed)abstract
- OBJECTIVES: : Epidemiological studies show that women with pregnancies complicated by hypertension have an increased risk of cardiovascular morbidity later in life. The underlying mechanisms to the risk increase remain largely unknown. This study evaluated sympathetic nerve activity in women with hypertensive pregnancies 40 years earlier compared to women with normotensive pregnancies. We hypothesized that sympathetic outflow would be increased in women with previous hypertensive pregnancies and that this partly may explain the increased cardiovascular risk. METHODS: : Sympathetic nerve activity to the muscle vascular bed [muscle sympathetic nerve activity (MSNA)] was recorded in 28 women, 18 with and 10 without a hypertensive manifestation during pregnancy. Women were also examined with ambulatory blood pressure measurements, pulse wave velocity, blood pressure response during Stroop test and laboratory analysis. RESULTS: : Women with previous hypertensive pregnancies did not show an increased sympathetic outflow compared to women with normotensive pregnancies. In eight women with treated hypertension sympathetic outflow was increased compared to normotensive women despite similar ambulatory blood pressure values (P < 0.05). During Stroop test the hypertensive women showed increased systolic blood pressure and also displayed the highest augmentation index compared to normotensive women (P < 0.05). CONCLUSION: : Hypertensive pregnancies per se were not associated with increased sympathetic outflow 40 years later. The increased cardiovascular risk in women with previous hypertensive pregnancies cannot be explained by chronic activation of the sympathetic nervous system.In women with previous hypertensive pregnancies, still hypertensive though well controlled, sympathetic outflow and arterial stiffness were, however, increased compared to normotensive counterparts.
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| 8. |
- Collén, Anna, et al.
(author)
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Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)phosphate aqueous two-phase system
- 2002
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In: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 943:1, s. 55-62
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Journal article (peer-reviewed)abstract
- Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)(2), (WP)(4) or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)(4) extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)(4) fused to the catalytic module and a short sequence of the linker [EGI(core-P5)(WP)(4)] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)(4) tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate.
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| 9. |
- Collén, Anna, et al.
(author)
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Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers
- 2001
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In: Journal of Biotechnology. - 1873-4863. ; 87:2, s. 179-191
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Journal article (peer-reviewed)abstract
- Endoglucanases (endo-1,4---glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide–propylene oxide random copolymer (EO50PO50) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)4 tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)2 tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)4 improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)2
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| 10. |
- Collén, Anna, et al.
(author)
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Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems - Effect of fusion localization on endoglucanase I of Trichoderma reesei
- 2001
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In: Journal of Chromatography A. - 0021-9673. ; 910:2, s. 275-284
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Journal article (peer-reviewed)abstract
- Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.
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