Publications by authors named "Maria Bloksgaard"

27 Publications

  • Page 1 of 1

Retinal vascular fractal dimensions and their association with macrovascular cardiac disease.

Ophthalmic Res 2021 Jan 15. Epub 2021 Jan 15.

Introduction As the only part of the human vasculature, retina is available for direct, non-invasive inspection. Retinal vascular fractal dimension (DF) is a method to measure the structure of the retinal vascular tree, with higher non-integer values between 1 and 2 representing a more complex and dense retinal vasculature. Retinal vascular structure has been associated with a variety of systemic diseases and this study examined the association of DF and macrovascular cardiac disease in a case-control design. Methods Retinal fundus photos were captured with Topcon TRC-50X in 38 persons that had coronary artery bypass grafting (CABG, cases) and 37 cardiovascular healthy controls. The semi-automatic software VAMPIRE was used to measure retinal DF. Results Patients with CABG had lower DF of the retinal main venular vessels compared to the control group (1.15 vs. 1.18, p=0.01). In a multivariable regression model adjusted for gender and age, eyes in the fourth quartile with higher DF were less likely to have CABG compared to patients in the first (OR, 7.20; 95% confidence interval, 1.63 to 31.86; p=0.009) and second quartile (OR, 8.25; 95% confidence interval, 1.70 to 40.01; p=0.009). Conclusions This study demonstrates that lower complexity of main venular vessels associates with higher risk of having CABG. The research supports the hypothesis that the retinal vascular structure can be used to assess non-ocular macrovascular disease.
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http://dx.doi.org/10.1159/000514442DOI Listing
January 2021

LIMK (LIM Kinase) Inhibition Prevents Vasoconstriction- and Hypertension-Induced Arterial Stiffening and Remodeling.

Hypertension 2020 Aug 29;76(2):393-403. Epub 2020 Jun 29.

From the Dalton Cardiovascular Research Center (M.M.-Q., F.I.R.-P., C.A.F., T.G., L.F.-S., C.M.-A., J.P., L.A.M.-L.), University of Missouri, Columbia, MO.

Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.15203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347448PMC
August 2020

Coronary artery bypass surgery independently associates with retinal vascular oxygen saturation.

Acta Ophthalmol 2020 Nov 17;98(7):709-715. Epub 2020 Apr 17.

Department of Ophthalmology, Odense University Hospital, Odense, Denmark.

Purpose: The retinal vasculature is the only part of the microcirculation that can be directly studied by non-invasive imaging. Based on the hypothesis that the systemic circulation is reflected in retinal vessels, we investigated if coronary artery bypass grafting (CABG) is related to changes in retinal vascular oxygen saturation (rSatO ).

Methods: Retinal metabolism was evaluated by Oxymap T1, which simultaneously captures two retinal images at different wavelengths measuring the retinal arteriolar (raSatO ) and venular (rvSatO ) oxygen saturation. Three to 4 days after surgery, we measured the median rSatO after CABG in 38 patients and in 39 healthy controls (operated for cataract).

Results: Coronary artery bypass grafting patients had higher raSatO (median ± standard deviation 93.1 ± 6.7% versus 90.5 ± 11.2%, p = 0.001) and rvSatO (57.4 ± 8.3% versus 53.5 ± 15.4%, p = 0.048) compared to healthy controls. In multivariable linear regression models, raSatO independently associated with CABG (coefficient + 3.6% in CABG patients, p = 0.007), and rvSatO correlated with gender (coefficient + 9.4% for females, p = 0.001) and CABG (coefficient + 8.2% in patients with CABG, p = 0.001).

Conclusions: Comparing patients with and without cardiovascular disease, raSatO and rvSatO positively and independently associated with CABG, suggesting their potential as non-invasive markers for coronary large artery disease.
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http://dx.doi.org/10.1111/aos.14444DOI Listing
November 2020

Implementing collaborative, active learning using peer instructions in pharmacology teaching increases students' learning and thereby exam performance.

Eur J Pharmacol 2020 Jan 14;867:172792. Epub 2019 Nov 14.

Institute of Molecular Medicine, Department of Cardiovascular and Renal Research, J. B. Winsløws Vej 21, 5000 Odense C, University of Southern Denmark, Denmark. Electronic address:

Teaching is a highly complex activity that draws on many kinds of knowledge. In this paper, we present our findings on the impact of implementing collaborative, active learning using peer instructions in pharmacology lectures. The hypothesis "implementation of collaborative, active learning increases students' understanding of pharmacological concepts, and thereby exam performance" was proven. Active learning was implemented in lectures on pharmacodynamics (10% of total lectures) in three different pharmacology courses. Teaching methods in the remaining lectures in the courses were unchanged. Prior to each lecture on pharmacodynamics, students were instructed to prepare using questions addressing key concepts in the curriculum that would be covered by the following lecture. In the lectures, students' understanding of these concepts was assessed using anonymous, online polls (ConcepTests). Collaborative learning was supported using peer instructions. Using the anonymous results of 15 written exams with 1097 submissions we show that the percentage of students achieving 50% or more of maximum points per exam question is significantly increased in questions relating to peer instructions lectures (P = 0.029, Odds Ratio (IQR) 1.83 (1.07, 3.15)). The observation is not explained by a general improvement of student performance (P = 0.289, Odds Ratio 1.15 (0.89, 1.51)). Both students' and the teacher have a positive perception of the active learning strategy and the possibility to give and receive instant feedback on students' learning progress. We conclude that implementation of collaborative, active learning using peer instructions, significantly improves students' learning in pharmacology.
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http://dx.doi.org/10.1016/j.ejphar.2019.172792DOI Listing
January 2020

Retinal vascular oxygen saturation increases after cardiac surgery.

Acta Ophthalmol 2019 Sep 7;97(6):e941-e942. Epub 2019 Mar 7.

Department of Ophthalmology, Odense University Hospital, Odense, Denmark.

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http://dx.doi.org/10.1111/aos.14084DOI Listing
September 2019

Local enrichment of fatty acid-binding protein 4 in the pericardial cavity of cardiovascular disease patients.

PLoS One 2018 5;13(11):e0206802. Epub 2018 Nov 5.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.

Background: The pericardial fluid may be representative of the interstitium of the heart. The aim of this study was to discriminate in cardiovascular disease patients between adipocytokines that are produced locally by the heart and those supplied by the circulation.

Methods: Enzyme-linked immunosorbent assays (ELISA) were used to determine levels of N-terminal pro-brain natriuretic peptide (NT-pBNP), fatty acid-binding protein 4 (FABP4), leptin, lipocalin-2, neutrophil elastase, proteinase-3, high sensitivity C-reactive protein (hsCRP) and adiponectin in venous plasma and pericardial fluid harvested during elective cardio-thoracic surgery (n = 132-152).

Results: In pericardial fluid compared to plasma, the levels were significantly smaller (p < 0.001) for leptin, lipocalin-2, neutrophil elastase, proteinase-3, hsCRP and adiponectin. For these biomarkers, the ratio of pericardial fluid-to-plasma level ([PF]/[P], median (interquartile range)) was 0.65 (0.47-1.01), 0.78 (0.56-1.09), 0.23 (0.11-0.60), 0.17 (0.09-0.36), 0.14 (0.08-0.35), and 0.25 (0.15-0.34), respectively. In contrast, pericardial fluid was significantly enriched (p < 0.001) in NT-pBNP ([PF]/[P]: 1.9 (1.06-2.73)) and even more so for FABP4 ([PF]/[P]: 3.90 (1.47-9.77)). Moreover, in pericardial fluid, the adipocytokines interrelated all significantly positive and correlated negative to hsCRP, whereas for NT-pBNP only a significantly positive correlation with adiponectin was found. These interrelations were distinct from those in the plasma, as were the correlations of the pericardial biomarkers with patient characteristics compared to plasma.

Conclusions: In cardiovascular disease patients, the pericardial cavity is a distinct adipocytokine microenvironment in which especially FABP4 is mainly derived from the heart.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206802PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218069PMC
April 2019

Extracellular matrix in cardiovascular pathophysiology.

Am J Physiol Heart Circ Physiol 2018 12 21;315(6):H1687-H1690. Epub 2018 Sep 21.

Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri , Columbia, Missouri.

The extracellular matrix (ECM) actively participates in diverse aspects of cardiovascular development and physiology as well as during disease development and progression. ECM roles are determined by its physical and mechanical properties and by its capacity to both release bioactive signals and activate cell signaling pathways. The ECM serves as a storage depot for a wide variety of molecules released in response to injury or with aging. Indeed, there is a plethora of examples describing how cells react to or modify ECM stiffness, how cells initiate intracellular signaling pathways, and how cells respond to the ECM. This Perspectives article reviews the contributions of 21 articles published in the American Journal of Physiology-Heart and Circulatory Physiology in response to a Call for Papers on this topic. Here, we summarize the contributions of these studies focused on the cardiac and vascular ECM. We highlight the translational importance of these studies and conclude that the ECM is a critical component of both the heart and vasculature. Readers are urged to examine and learn from this special Call for Papers.
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http://dx.doi.org/10.1152/ajpheart.00631.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336969PMC
December 2018

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy.

J Vis Exp 2018 04 9(134). Epub 2018 Apr 9.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark; Department of Cardiac, Thoracic and Vascular Surgery, Odense University Hospital.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.
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http://dx.doi.org/10.3791/57451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933429PMC
April 2018

Relaxing Responses to Hydrogen Peroxide and Nitric Oxide in Human Pericardial Resistance Arteries Stimulated with Endothelin-1.

Basic Clin Pharmacol Toxicol 2018 Jan 30;122(1):74-81. Epub 2017 Jul 30.

Department of Cardiovascular and Renal Research, Centre for Individualized Medicine in Arterial Diseases (CIMA), Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

In human pericardial resistance arteries, effects of the endothelium-dependent vasodilator bradykinin are mediated by NO during contraction induced by K or the TxA analogue U46619 and by H O during contraction by endothelin-1 (ET-1), respectively. We tested the hypotheses that ET-1 reduces relaxing effects of NO and increases those of H O in resistance artery smooth muscle of patients with cardiovascular disease. Arterial segments, dissected from the parietal pericardium of 39 cardiothoracic surgery patients, were studied by myography during amplitude-matched contractions induced by K , the TXA analogue U46619 or ET-1. Effects of the NO donor Na-nitroprusside (SNP) and of exogenous H O were recorded in the absence and presence of inhibitors of cyclooxygenases, NO synthases and small and intermediate conductance calcium-activated K channels. During contractions induced by either of the three stimuli, the potency of SNP did not differ and was not modified by the inhibitors. In vessels contracted with ET-1, the potency of H O was on average and in terms of interindividual variability considerably larger than in K -contracted vessels. Both differences were not statistically significant in the presence of inhibitors of mechanisms of endothelium-dependent vasodilatation. In resistance arteries from patients with cardiovascular disease, ET-1 does not selectively modify smooth muscle relaxing responses to NO or H O . Furthermore, the candidate endothelium-derived relaxing factor H O also acts as an endothelium-dependent vasodilator.
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http://dx.doi.org/10.1111/bcpt.12843DOI Listing
January 2018

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

Am J Physiol Heart Circ Physiol 2017 Jul 21;313(1):H164-H178. Epub 2017 Apr 21.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies. This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.
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http://dx.doi.org/10.1152/ajpheart.00110.2017DOI Listing
July 2017

Endothelial SIRT1 prevents adverse arterial remodeling by facilitating HERC2-mediated degradation of acetylated LKB1.

Oncotarget 2016 Jun;7(26):39065-39081

State Key Laboratory of Pharmaceutical Biotechnology and Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China.

Aims-SIRT1 exerts potent activity against cellular senescence and vascular ageing. By decreasing LKB1 protein levels, it promotes the survival and regeneration of endothelial cells. The present study aims to investigate the molecular mechanisms underlying SIRT1-mediated LKB1 degradation for the prevention of vascular ageing.Methods and Results-Co-immunoprecipitation assay demonstrated that SIRT1, via its amino-terminus, binds to the DOC domain of HERC2 [HECT and RLD domain containing E3 ubiquitin protein ligase 2], which then ubiquitinates LKB1 in the nuclear compartment of endothelial cells. Site-directed mutagenesis revealed that acetylation at lysine (K) 64 of LKB1 triggers the formation of SIRT1/HERC2/LKB1 protein complex and subsequent proteasomal degradation. In vitro cellular studies suggested that accumulation of acetylated LKB1 in the nucleus leads to endothelial activation, in turn stimulating the proliferation of vascular smooth muscle cells and the production of extracellular matrix proteins. Chromatin immunoprecipitation quantitative PCR confirmed that acetylated LKB1 interacts with and activates TGFβ1 promoter, which is inhibited by SIRT1. Knocking down either SIRT1 or HERC2 results in an increased association of LKB1 with the positive regulatory elements of TGFβ1 promoter. In mice without endothelial nitric oxide synthase, selective overexpression of human SIRT1 in endothelium prevents hypertension and age-related adverse arterial remodeling. Lentiviral-mediated knockdown of HERC2 abolishes the beneficial effects of endothelial SIRT1 on both arterial remodeling and arterial blood pressure control.Conclusion-By downregulating acetylated LKB1 protein via HERC2, SIRT1 fine-tunes the crosstalk between endothelial and vascular smooth muscle cells to prevent adverse arterial remodeling and maintain vascular homeostasis.
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http://dx.doi.org/10.18632/oncotarget.9687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129914PMC
June 2016

Biochemical and Bioimaging Evidence of Cholesterol in Acquired Cholesteatoma.

Ann Otol Rhinol Laryngol 2016 Aug 15;125(8):627-33. Epub 2016 Apr 15.

MEMPHYS-Centre for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark

Objectives: To quantify the barrier sterols and image the lipid structures in the matrix of acquired cholesteatoma and compare the distribution with that found in stratum corneum from normal skin, with the goal to resolve their potential influence on cholesteatoma growth.

Methods: High-performance thin-layer chromatography (HPTLC) was used to achieve a quantitative biochemical determination of the sterols. The intercellular lipids were visualized by Coherent Anti-Stokes Raman scattering (CARS) microscopy, which enables label-free imaging of the lipids in intact tissue samples.

Results: The results show that the total lipid content of the cholesteatoma matrix is similar to that of stratum corneum from skin and that the cholesteatoma matrix unquestionably contains cholesterol. The cholesterol content in the cholesteatoma matrix is increased by over 30% (w/w dry weight) compared to the control. The cholesterol sulfate content is below 1% of the total lipids in both the cholesteatoma and the control. Cholesterol ester was reduced by over 30% when compared to the control.

Conclusions: The content of cholesterol in the cholesteatoma matrix is significantly different from that in stratum corneum from skin, and we confirm that the main structure of the cholesteatoma resembles very thick stratum corneum.
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http://dx.doi.org/10.1177/0003489416642784DOI Listing
August 2016

Endothelin-1 shifts the mediator of bradykinin-induced relaxation from NO to H2 O2 in resistance arteries from patients with cardiovascular disease.

Br J Pharmacol 2016 05 6;173(10):1653-64. Epub 2016 Apr 6.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Background And Purpose: We tested the hypothesis that in resistance arteries from cardiovascular disease (CVD) patients, effects of an endothelium-dependent vasodilator depend on the contractile stimulus.

Experimental Approach: Arteries dissected from parietal pericardium of cardiothoracic surgery patients were studied by myography and imaging techniques. Segments were sub-maximally contracted by K(+) , the TxA2 analogue U46619 or endothelin-1 (ET-1).

Key Results: Relaxing effects of Na-nitroprusside were comparable, but those of bradykinin (BK) were bigger in the presence of ET-1 compared with K(+) or U46619. BK-induced relaxation was (i) abolished by L-NAME in K(+) -contracted arteries, (ii) partly inhibited by L-NAME in the presence of U46619 and (iii) not altered by indomethacin, L-NAME plus inhibitors of small and intermediate conductance calcium-activated K(+) channels, but attenuated by catalase, in ET-1-contracted arteries. This catalase-sensitive relaxation was unaffected by inhibitors of NADPH oxidases or allopurinol. Exogenous H2 O2 caused a larger relaxation of ET-1-induced contractions than those evoked by K(+) or U46619 in the presence of inhibitors of other endothelium-derived relaxing factors. Catalase-sensitive staining of cellular ROS with CellROX Deep Red was significantly increased in the presence of both 1 μM BK and 2 nM ET-1 but not either peptide alone.

Conclusions And Implications: In resistance arteries from patients with CVD, exogenous ET-1 shifts the mediator of relaxing responses to the endothelium-dependent vasodilator BK from NO to H2 O2 and neither NADPH oxidases, xanthine oxidase nor NOS appear to be involved in this effect. This might have consequences for endothelial dysfunction in conditions where intra-arterial levels of ET-1 are enhanced.
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http://dx.doi.org/10.1111/bph.13467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842913PMC
May 2016

Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP-/- mice.

J Lipid Res 2015 Sep 4;56(9):1738-46. Epub 2015 Jul 4.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.

Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene in the epidermis recapitulate this phenotype, whereas generation of an artificial epidermal barrier during gland development reverses the phenotype. Our findings indicate that the Harderian gland is activated by the compromised epidermal barrier as an adaptive and protective mechanism to overcome the barrier defect.
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http://dx.doi.org/10.1194/jlr.M060780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548778PMC
September 2015

Elastin organization in pig and cardiovascular disease patients' pericardial resistance arteries.

J Vasc Res 2015 31;52(1):1-11. Epub 2015 Mar 31.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Peripheral vascular resistance is increased in essential hypertension. This involves structural changes of resistance arteries and stiffening of the arterial wall, including remodeling of the extracellular matrix. We hypothesized that biopsies of the human parietal pericardium, obtained during coronary artery bypass grafting or cardiac valve replacement surgeries, can serve as a source of resistance arteries for structural research in cardiovascular disease patients. We applied two-photon excitation fluorescence microscopy to study the parietal pericardium and isolated pericardial resistance arteries with a focus on the collagen and elastin components of the extracellular matrix. Initial findings in pig tissue were confirmed in patient biopsies. The microarchitecture of the internal elastic lamina in both the pig and patient pericardial resistance arteries (studied at a transmural pressure of 100 mm Hg) is fiber like, and no prominent external elastic lamina could be observed. This microarchitecture is very different from that in rat mesenteric arteries frequently used for resistance artery research. In conclusion, we add three-dimensional information on the structure of the extracellular matrix in resistance arteries from cardiovascular disease patients and propose further use of patient pericardial resistance arteries for studies of the human microvasculature.
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http://dx.doi.org/10.1159/000376548DOI Listing
July 2015

Delayed hepatic adaptation to weaning in ACBP-/- mice is caused by disruption of the epidermal barrier.

Cell Rep 2013 Dec 5;5(5):1403-12. Epub 2013 Dec 5.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark. Electronic address:

We previously reported that mice deficient in acyl-CoA-binding protein (ACBP) display a delayed metabolic adaptation to weaning. This includes a delayed activation of the hepatic lipogenic gene program, which may result from hepatic accumulation of triacylglycerol and/or cholesteryl esters in the late suckling period. To further investigate the basis for this phenotype, we generated mice deficient in ACBP in hepatocytes (Alb-ACBP(-/-)) and keratinocytes (K14-ACBP(-/-)). Surprisingly, the delayed adaptation to weaning, including hepatic lipid accumulation, is caused by ACBP deficiency in the skin rather than in the liver. Similarly to ACBP(-/-) mice, K14-ACBP(-/-) mice exhibit an increased transepidermal water loss, and we show that the hepatic phenotype is caused specifically by the epidermal barrier defect, which leads to increased lipolysis in white adipose tissue. Our data demonstrate that an imperfect epidermal barrier leads to profound suppression of the hepatic SREBP gene program and lipid accumulation in the liver.
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http://dx.doi.org/10.1016/j.celrep.2013.11.010DOI Listing
December 2013

Acyl-CoA binding protein and epidermal barrier function.

Biochim Biophys Acta 2014 Mar 29;1841(3):369-76. Epub 2013 Sep 29.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark. Electronic address:

The acyl-CoA binding protein (ACBP) is a 10kDa intracellular protein expressed in all eukaryotic species and mammalian tissues investigated. It binds acyl-CoA esters with high specificity and affinity and is thought to act as an intracellular transporter of acyl-CoA esters between different enzymatic systems; however, the precise function remains unknown. ACBP is expressed at relatively high levels in the epidermis, particularly in the suprabasal layers, which are highly active in lipid synthesis. Targeted disruption of the ACBP gene in mice leads to a pronounced skin and fur phenotype, which includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced levels of non-esterified very long chain fatty acids in the stratum corneum of ACBP(-/-) mice. Here we review the current knowledge of ACBP with special focus on the function of ACBP in the epidermal barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.
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http://dx.doi.org/10.1016/j.bbalip.2013.09.013DOI Listing
March 2014

Structural and dynamical aspects of skin studied by multiphoton excitation fluorescence microscopy-based methods.

Eur J Pharm Sci 2013 Dec 19;50(5):586-94. Epub 2013 Apr 19.

Membrane Biophysics and Biophotonics group/MEMPHYS, Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes' parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe--2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) and diffusion coefficients of distinct fluorescence probes (raster imaging correlation spectroscopy) can be obtained from different regions of the tissue. Comparative studies of different tissue strata, but also between equivalent regions of normal and abnormal excised skin, including applications of fluctuation correlation spectroscopy on transdermal penetration of liposomes are presented and discussed. The data from the different studies reported reveal the intrinsic heterogeneity of skin and also prove these strategies to be powerful noninvasive tools to explore structural and dynamical aspects of the tissue.
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http://dx.doi.org/10.1016/j.ejps.2013.04.010DOI Listing
December 2013

Spatially resolved two-color diffusion measurements in human skin applied to transdermal liposome penetration.

J Invest Dermatol 2013 May 6;133(5):1260-8. Epub 2012 Dec 6.

Membrane Biophysics and Biophotonics Group/MEMPHYS Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

A multiphoton excitation-based fluorescence fluctuation spectroscopy method, Raster image correlation spectroscopy (RICS), was used to measure the local diffusion coefficients of distinct model fluorescent substances in excised human skin. In combination with structural information obtained by multiphoton excitation fluorescence microscopy imaging, the acquired diffusion information was processed to construct spatially resolved diffusion maps at different depths of the stratum corneum (SC). Experiments using amphiphilic and hydrophilic fluorescently labeled molecules show that their diffusion in SC is very heterogeneous on a microscopic scale. This diffusion-based strategy was further exploited to investigate the integrity of liposomes during transdermal penetration. Specifically, the diffusion of dual-color fluorescently labeled liposomes--containing an amphiphilic fluorophore in the lipid bilayer and a hydrophilic fluorophore encapsulated in the liposome lumen--was measured using cross-correlation RICS. This type of experiment allows discrimination between separate (uncorrelated) and joint (correlated) diffusion of the two different fluorescent probes, giving information about liposome integrity. Independent of the liposome composition (phospholipids or transfersomes), our results show a clear lack of cross-correlation below the skin surface, indicating that the penetration of intact liposomes is highly compromised by the skin barrier.
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http://dx.doi.org/10.1038/jid.2012.461DOI Listing
May 2013

The acyl-CoA binding protein is required for normal epidermal barrier function in mice.

J Lipid Res 2012 Oct 24;53(10):2162-74. Epub 2012 Jul 24.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.
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http://dx.doi.org/10.1194/jlr.M029553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435549PMC
October 2012

The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.

J Invest Dermatol 2012 Sep 26;132(9):2215-25. Epub 2012 Apr 26.

Department of Cell and Molecular Biology (CMB), Karolinska Institutet, Stockholm, Sweden.

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skin's surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skin's lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skin's low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barrier's robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.
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http://dx.doi.org/10.1038/jid.2012.43DOI Listing
September 2012

Structural characterization and lipid composition of acquired cholesteatoma: a comparative study with normal skin.

Otol Neurotol 2012 Feb;33(2):177-83

Membrane Biophysics and Biophotonics Group/MEMPHYS-Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.

Hypothesis: The goal of this work is to characterize the morphology and lipid composition of acquired cholesteatoma. We hypothesize that constitutive lipid membranes are present in the cholesteatoma and resemble those found in human skin stratum corneum.

Methods: We performed a comparative noninvasive structural and lipid compositional study of acquired cholesteatoma and control human skin using multiphoton excitation fluorescence microscopy-related techniques and high-performance thin-layer chromatography.

Results: The structural arrangement of the cholesteatoma is morphologically invariant along a depth of more than 200 μm and resembles the stratum corneum of hyperorthokeratotic skin. Lipid compositional analyses of the cholesteatoma show the presence of all major lipid classes found in normal skin stratum corneum (ceramides, long chain fatty acids, and cholesterol). Consistent with this, evaluation of Nile red and LAURDAN generalized polarization function images of the cholesteatoma show intercellular regions similar to normal skin stratum corneum in terms of lipid membrane packing and local water content.

Conclusion: The investigations show the presence of an extremely thickened stratum corneum within the cholesteatoma. The lipid composition and extracellular membranes similar to those of normal skin stratum corneum are present, indicating that a defensive/permeability barrier is present in the cholesteatoma. Finally, it is demonstrated that multiphoton excitation fluorescence microscopy is a suitable noninvasive tool for investigating the morphology and intrinsic physical properties of acquired cholesteatoma.
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http://dx.doi.org/10.1097/MAO.0b013e318241be63DOI Listing
February 2012

Mice with targeted disruption of the acyl-CoA binding protein display attenuated urine concentrating ability and diminished renal aquaporin-3 abundance.

Am J Physiol Renal Physiol 2012 Apr 11;302(8):F1034-44. Epub 2012 Jan 11.

Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, Denmark.

The acyl-CoA binding protein (ACBP) is a small intracellular protein that specifically binds and transports medium to long-chain acyl-CoA esters. Previous studies have shown that ACBP is ubiquitously expressed but found at particularly high levels in lipogenic cell types as well as in many epithelial cells. Here we show that ACBP is widely expressed in human and mouse kidney epithelium, with the highest expression in the proximal convoluted tubules. To elucidate the role of ACBP in the renal epithelium, mice with targeted disruption of the ACBP gene (ACBP(-/-)) were used to study water and NaCl balance as well as urine concentrating ability in metabolic cages. Food intake and urinary excretion of Na(+) and K(+) did not differ between ACBP(-/-) and (+/+) mice. Interestingly, however, water intake and diuresis were significantly higher at baseline in ACBP(-/-) mice compared with that of (+/+) mice. Subsequent to 20-h water deprivation, ACBP(-/-) mice exhibited increased diuresis, reduced urine osmolality, elevated hematocrit, and higher relative weight loss compared with (+/+) mice. There were no significant differences in plasma concentrations of renin, corticosterone, and aldosterone between mice of the two genotypes. After water deprivation, renal medullary interstitial fluid osmolality and concentrations of Na(+), K(+), and urea did not differ between genotypes and cAMP excretion was similar. Renal aquaporin-1 (AQP1), -2, and -4 protein abundances did not differ between water-deprived (+/+) and ACBP(-/-) mice; however, ACBP(-/-) mice displayed increased apical targeting of pS256-AQP2. AQP3 abundance was lower in ACBP(-/-) mice than in (+/+) control animals. Thus we conclude that ACBP is necessary for intact urine concentrating ability. Our data suggest that the deficiency in urine concentrating ability in the ACBP(-/-) may be caused by reduced AQP3, leading to impaired efflux over the basolateral membrane of the collecting duct.
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http://dx.doi.org/10.1152/ajprenal.00371.2011DOI Listing
April 2012

Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.

J Biol Chem 2011 Feb 24;286(5):3460-72. Epub 2010 Nov 24.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.

The acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor is an intracellular protein that binds C(14)-C(22) acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP(-/-)). These mice are viable and fertile and develop normally. However, around weaning, the ACBP(-/-) mice go through a crisis with overall weakness and a slightly decreased growth rate. Using microarray analysis, we show that the liver of ACBP(-/-) mice displays a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element-binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors, leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to delayed induction of the lipogenic gene program in the liver.
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http://dx.doi.org/10.1074/jbc.M110.161109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030352PMC
February 2011

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis.

J Biol Chem 2009 Jan 17;284(2):921-9. Epub 2008 Nov 17.

Department of Cell Physiology and Metabolism, Geneva, Switzerland.

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In betaGlud1(-/-) mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.
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http://dx.doi.org/10.1074/jbc.M806295200DOI Listing
January 2009

The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes.

J Biol Chem 2005 Feb 15;280(7):5258-66. Epub 2004 Dec 15.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular lipid-binding protein that transports acylCoA esters. The protein is expressed in most cell types at low levels; however, expression is particularly high in cells with a high turnover of fatty acids. Here we confirm a previous observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target gene in hepatocytes. Members of the SREBP family activate the rat ACBP gene through binding sites for SREBP and the auxiliary factors Sp1 and nuclear factor Y in the proximal promoter. In addition, we show that ACBP is a peroxisome proliferator-activated receptor (PPAR) alpha target gene in cultured hepatocytes and is induced in the liver by fibrates in a PPARalpha-dependent manner. Thus, ACBP is a dual PPARalpha and SREBP-1c target gene in hepatocytes. Fasting leads to reduced activity of SREBP but increased activity of PPARalpha in hepatocytes, and in keeping with ACBP being a dual target gene, we show that ACBP expression is significantly lower in livers from PPARalpha knock-out mice than in livers from wild type mice. In conclusion, expression of ACBP in rodent hepatocytes is subject to dual metabolic regulation by PPARalpha and SREBP-1c, which may reflect the need for ACBP during lipogenic as well as lipo-oxidative conditions.
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http://dx.doi.org/10.1074/jbc.M407515200DOI Listing
February 2005