Setting up an institutional OMERO environment for bioimage data: Perspectives from both facility staff and users.

Modern bioimaging core facilities at research institutions are essential for managing and maintaining high-end instruments, providing training and support for researchers in experimental design, image acquisition and data analysis. An important task for these facilities is the professional management of complex multidimensional bioimaging data, which are often produced in large quantity and very different file formats. This article details the process that led to successfully implementing the OME Remote Objects system (OMERO) for bioimage-specific research data management (RDM) at the Core Facility Cellular Imaging (CFCI) at the Technische Universität Dresden (TU Dresden). Ensuring compliance with the FAIR (findable, accessible, interoperable, reusable) principles, we outline here the challenges that we faced in adapting data handling and storage to a new RDM system. These challenges included the introduction of a standardised group-specific naming convention, metadata curation with tagging and Key-Value pairs, and integration of existing image processing workflows. By sharing our experiences, this article aims to provide insights and recommendations for both individual researchers and educational institutions intending to implement OMERO as a management system for bioimaging data. We showcase how tailored decisions and structured approaches lead to successful outcomes in RDM practices.

Oxygenator assisted dynamic microphysiological culture elucidates the impact of hypoxia on valvular interstitial cell calcification.

INTRODUCTION: Microphysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation. METHODS: Human valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts. RESULTS: Hypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h. CONCLUSION: In hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Tissue requirements for the application of aortic valve neocuspidization - appropriate pericardium properties and homogeneity?

OBJECTIVE: Aortic valve neocuspidization (AVNeo) using autologous pericardium is a promising technique. Expected advantages are reduced immune response, appropriate biomechanics and lower treatment expenses. Nevertheless, autologous pericardium can be affected by patient's age and comorbidities. Usually, glutaraldehyde (GA) - fixed bovine pericardium is the basic material for aortic valve prostheses, easy available and carefully pre-examined in a standardized fabrication process. Aim of the study is the verification of autologous pericardial tissue homogeneity by analysing tissue thickness, biomechanics and extracellular matrix (ECM) composition. METHODS: Segments of human GA-fixed pericardium selected by the surgeon based on visual criteria for cusp pre-cut and remaining after surgical AV replacement were investigated in comparison to bovine standard tissue treated equivalently. Pericardium sampling was performed at up to three positions of each sutured cusp for histological or biomechanical analysis, according to tissue availability. RESULTS AND CONCLUSIONS: Human pericardia exhibited a higher heterogeneity in collagen content, density of vessel structures and elastic moduli. Thickness, vessel density and collagen and elastin content differed significantly between the species. In contrast, significant interindividual differences were detected in most properties investigated for human pericardial samples but only for tissue thickness in bovine tissues. Higher heterogeneity of human pericardium, differing vessel and collagen content compared to bovine state-of-the-art material might be detrimental for long term AV functionality or deterioration and have to be intensely investigated in patients follow up after autologous cusp replacement.

Challenges of aortic valve tissue culture - maintenance of viability and extracellular matrix in the pulsatile dynamic microphysiological system.

BACKGROUND: Calcific aortic valve disease (CAVD) causes an increasing health burden in the 21st century due to aging population. The complex pathophysiology remains to be understood to develop novel prevention and treatment strategies. Microphysiological systems (MPSs), also known as organ-on-chip or lab-on-a-chip systems, proved promising in bridging in vitro and in vivo approaches by applying integer AV tissue and modelling biomechanical microenvironment. This study introduces a novel MPS comprising different micropumps in conjunction with a tissue-incubation-chamber (TIC) for long-term porcine and human AV incubation (pAV, hAV). RESULTS: Tissue cultures in two different MPS setups were compared and validated by a bimodal viability analysis and extracellular matrix transformation assessment. The MPS-TIC conjunction proved applicable for incubation periods of 14-26 days. An increased metabolic rate was detected for pulsatile dynamic MPS culture compared to static condition indicated by increased LDH intensity. ECM changes such as an increase of collagen fibre content in line with tissue contraction and mass reduction, also observed in early CAVD, were detected in MPS-TIC culture, as well as an increase of collagen fibre content. Glycosaminoglycans remained stable, no significant alterations of α-SMA or CD31 epitopes and no accumulation of calciumhydroxyapatite were observed after 14 days of incubation. CONCLUSIONS: The presented ex vivo MPS allows long-term AV tissue incubation and will be adopted for future investigation of CAVD pathophysiology, also implementing human tissues. The bimodal viability assessment and ECM analyses approve reliability of ex vivo CAVD investigation and comparability of parallel tissue segments with different treatment strategies regarding the AV (patho)physiology.

Hemocompatibility tuning of an innovative glutaraldehyde-free preparation strategy using riboflavin/UV crosslinking and electron irradiation of bovine pericardium for cardiac substitutes.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.

Infrared Spectroscopic Verification of a α-Helical Collagen Structure in Glutaraldehyde-Free Crosslinked Bovine Pericardium for Cardiac Implants.

The degeneration of heart valve bioprostheses due to calcification processes is caused by the intercalation of calciumhydroxyapatite in pericardium collagen bundles. Variations of the protein secondary structure of biomaterials according to preparation are relevant for this mineralization process and thus the structural characterization of innovative bioprostheses materials is of great importance. The gold standard for prostheses preparation is glutaraldehyde (GA)-fixation of bovine pericardium that adversely promotes calcification. The novel GA-free SULEEI-treatment of bovine pericardium includes decellularization, UV-crosslinking, and electron beam sterilization. The aim of this study is the structural characterization of SULEEI-treated and GA-fixed bovine pericardium. IR spectroscopic imaging combined with multivariate data and curve fit analysis was applied to investigate the amide I and amide II regions of SULEEI-treated and GA-fixed samples. The spectroscopic images of GA-fixed pericardial tissue exhibited a generally high content of amine groups and side chains providing nucleation points for calcification processes. In contrast, in SULEEI-treated tissue, the typical α-helical structure was retained and was supposed to be less prone to deterioration.

Empagliflozin Preserves Skeletal Muscle Function in a HFpEF Rat Model.

Besides structural alterations in the myocardium, heart failure with preserved ejection fraction (HFpEF) is also associated with molecular and physiological alterations of the peripheral skeletal muscles (SKM) contributing to exercise intolerance often seen in HFpEF patients. Recently, the use of Sodium-Glucose-Transporter 2 inhibitors (SGLT2i) in clinical studies provided evidence for a significant reduction in the combined risk of cardiovascular death or hospitalization for HFpEF. The present study aimed to further elucidate the impact of Empagliflozin (Empa) on: (1) SKM function and metabolism and (2) mitochondrial function in an established HFpEF rat model. At the age of 24 weeks, obese ZSF1 rats were randomized either receiving standard care or Empa in the drinking water. ZSF1 lean animals served as healthy controls. After 8 weeks of treatment, echocardiography and SKM contractility were performed. Mitochondrial function was assessed in saponin skinned fibers and SKM tissue was snap frozen for molecular analyses. HFpEF was evident in the obese animals when compared to lean-increased E/é and preserved left ventricular ejection fraction. Empa treatment significantly improved E/é and resulted in improved SKM contractility with reduced intramuscular lipid content. Better mitochondrial function (mainly in complex IV) with only minor modulation of atrophy-related proteins was seen after Empa treatment. The results clearly documented a beneficial effect of Empa on SKM function in the present HFpEF model. These effects were accompanied by positive effects on mitochondrial function possibly modulating SKM function.

Targeting MuRF1 by small molecules in a HFpEF rat model improves myocardial diastolic function and skeletal muscle contractility.

BACKGROUND: About half of heart failure (HF) patients, while having preserved left ventricular function, suffer from diastolic dysfunction (so-called HFpEF). No specific therapeutics are available for HFpEF in contrast to HF where reduced ejection fractions (HFrEF) can be treated pharmacologically. Myocardial titin filament stiffening, endothelial dysfunction, and skeletal muscle (SKM) myopathy are suspected to contribute to HFpEF genesis. We previously described small molecules interfering with MuRF1 target recognition thereby attenuating SKM myopathy and dysfunction in HFrEF animal models. The aim of the present study was to test the efficacy of one small molecule (MyoMed-205) in HFpEF and to describe molecular changes elicited by MyoMed-205. METHODS: Twenty-week-old female obese ZSF1 rats received the MuRF1 inhibitor MyoMed-205 for 12 weeks; a comparison was made to age-matched untreated ZSF1-lean (healthy) and obese rats as controls. LV (left ventricle) function was assessed by echocardiography and by invasive haemodynamic measurements until week 32. At week 32, SKM and endothelial functions were measured and tissues collected for molecular analyses. Proteome-wide analysis followed by WBs and RT-PCR was applied to identify specific genes and affected molecular pathways. MuRF1 knockout mice (MuRF1-KO) SKM tissues were included to validate MuRF1-specificity. RESULTS: By week 32, untreated obese rats had normal LV ejection fraction but augmented E/e' ratios and increased end diastolic pressure and myocardial fibrosis, all typical features of HFpEF. Furthermore, SKM myopathy (both atrophy and force loss) and endothelial dysfunction were detected. In contrast, MyoMed-205 treated rats had markedly improved diastolic function, less myocardial fibrosis, reduced SKM myopathy, and increased SKM function. SKM extracts from MyoMed-205 treated rats had reduced MuRF1 content and lowered total muscle protein ubiquitination. In addition, proteomic profiling identified eight proteins to respond specifically to MyoMed-205 treatment. Five out of these eight proteins are involved in mitochondrial metabolism, dynamics, or autophagy. Consistent with the mitochondria being a MyoMed-205 target, the synthesis of mitochondrial respiratory chain complexes I + II was increased in treated rats. MuRF1-KO SKM controls also had elevated mitochondrial complex I and II activities, also suggesting mitochondrial activity regulation by MuRF1. CONCLUSIONS: MyoMed-205 improved myocardial diastolic function and prevented SKM atrophy/function in the ZSF1 animal model of HFpEF. Mechanistically, SKM benefited from an attenuated ubiquitin proteasome system and augmented synthesis/activity of proteins of the mitochondrial respiratory chain while the myocardium seemed to benefit from reduced titin modifications and fibrosis.

Use of MALDI Mass Spectrometry Imaging to Identify Proteomic Signatures in Aortic Aneurysms after Endovascular Repair.

Endovascular repair (EVAR) has become the standard procedure in treating thoracic (TAA) or abdominal aortic aneurysms (AAA). Not entirely free of complications, a persisting perfusion of the aneurysm after EVAR, called Endoleak (EL), leads to reintervention and risk of secondary rupture. How the aortic wall responds to the implantation of a stentgraft and EL is mostly uncertain. We present a pilot study to identify peptide signatures and gain new insights in pathophysiological alterations of the aortic wall after EVAR using matrix-assisted laser desorption or ionization mass spectrometry imaging (MALDI-MSI). In course of or accompanying an open aortic repair, tissue sections from 15 patients (TAA = 5, AAA = 5, EVAR = 5) were collected. Regions of interest (tunica media and tunica adventitia) were defined and univariate (receiver operating characteristic analysis) statistical analysis for subgroup comparison was used. This proof-of-concept study demonstrates that MALDI-MSI is feasible to identify discriminatory peptide signatures separating TAA, AAA and EVAR. Decreased intensity distributions for actin, tropomyosin, and troponin after EVAR suggest impaired contractility in vascular smooth muscle cells. Furthermore, inability to provide energy caused by impaired respiratory chain function and continuous degradation of extracellular matrix components (collagen) might support aortic wall destabilization. In case of EL after EVAR, this mechanism may result in a weakened aortic wall with lacking ability to react on reinstating pulsatile blood flow.

Brillouin confocal microscopy to determine biomechanical properties of SULEEI-treated bovine pericardium for application in cardiac surgery.

BACKGROUND: Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. OBJECTIVE: BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. METHODS: Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young's modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). RESULTS: SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young's modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. CONCLUSION: BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Sacubitril/Valsartan Improves Diastolic Function But Not Skeletal Muscle Function in a Rat Model of HFpEF.

The angiotensin receptor/neprilysin inhibitor Sacubitril/Valsartan (Sac/Val) has been shown to be beneficial in patients suffering from heart failure with reduced ejection fraction (HFrEF). However, the impact of Sac/Val in patients presenting with heart failure with preserved ejection fraction (HFpEF) is not yet clearly resolved. The present study aimed to reveal the influence of the drug on the functionality of the myocardium, the skeletal muscle, and the vasculature in a rat model of HFpEF. Female obese ZSF-1 rats received Sac/Val as a daily oral gavage for 12 weeks. Left ventricle (LV) function was assessed every four weeks using echocardiography. Prior to organ removal, invasive hemodynamic measurements were performed in both ventricles. Vascular function of the carotid artery and skeletal muscle function were monitored. Sac/Val treatment reduced E/é ratios, left ventricular end diastolic pressure (LVEDP) and myocardial stiffness as well as myocardial fibrosis and heart weight compared to the obese control group. Sac/Val slightly improved endothelial function in the carotid artery but had no impact on skeletal muscle function. Our results demonstrate striking effects of Sac/Val on the myocardial structure and function in a rat model of HFpEF. While vasodilation was slightly improved, functionality of the skeletal muscle remained unaffected.

Optical coherence tomography and multiphoton microscopy offer new options for the quantification of fibrotic aortic valve disease in ApoE-/- mice.

Aortic valve sclerosis is characterized as the thickening of the aortic valve without obstruction of the left ventricular outflow. It has a prevalence of 30% in people over 65 years old. Aortic valve sclerosis represents a cardiovascular risk marker because it may progress to moderate or severe aortic valve stenosis. Thus, the early recognition and management of aortic valve sclerosis are of cardinal importance. We examined the aortic valve geometry and structure from healthy C57Bl6 wild type and age-matched hyperlipidemic ApoE-/- mice with aortic valve sclerosis using optical coherence tomography (OCT) and multiphoton microscopy (MPM) and compared results with histological analyses. Early fibrotic thickening, especially in the tip region of the native aortic valve leaflets from the ApoE-/- mice, was detectable in a precise spatial resolution using OCT. Evaluation of the second harmonic generation signal using MPM demonstrated that collagen content decreased in all aortic valve leaflet regions in the ApoE-/- mice. Lipid droplets and cholesterol crystals were detected using coherent anti-Stokes Raman scattering in the tissue from the ApoE-/- mice. Here, we demonstrated that OCT and MPM, which are fast and precise contactless imaging approaches, are suitable for defining early morphological and structural alterations of sclerotic murine aortic valves.

ZSF1 rat as animal model for HFpEF: Development of reduced diastolic function and skeletal muscle dysfunction.

AIMS: The prevalence of heart failure with preserved ejection fraction (HFpEF) is still increasing, and so far, no pharmaceutical treatment has proven to be effective. A key obstacle for testing new pharmaceutical substances is the availability of suitable animal models for HFpEF, which realistically reflect the clinical picture. The aim of the present study was to characterize the development of HFpEF and skeletal muscle (SM) dysfunction in ZSF1 rats over time. METHODS AND RESULTS: Echocardiography and functional analyses of the SM were performed in 6-, 10-, 15-, 20-, and 32-week-old ZSF1-lean and ZSF1-obese. Furthermore, myocardial and SM tissue was collected for molecular and histological analyses. HFpEF markers were evident as early as 10 weeks of age. Diastolic dysfunction, confirmed by a significant increase in E/e', was detectable at 10 weeks. Increased left ventricular mRNA expression of collagen and BNP was detected in ZSF1-obese animals as early as 15 and 20 weeks, respectively. The loss of muscle force was measurable in the extensor digitorum longus starting at 15 weeks, whereas the soleus muscle function was impaired at Week 32. In addition, at Week 20, markers for aortic valve sclerosis were increased. CONCLUSIONS: Our measurements confirmed the appearance of HFpEF in ZSF1-obese rats as early as 10 weeks of age, most likely as a result of the pre-existing co-morbidities. In addition, SM function was reduced after the manifestation of HFpEF. In conclusion, the ZSF1 rat may serve as a suitable animal model to study pharmaceutical strategies for the treatment of HFpEF.

Treatment with XAV-939 prevents in vitro calcification of human valvular interstitial cells.

The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the ß-catenin complex and dephosphorylation of ß-catenin. The dephosphorylated active form of ß-catenin (non-phospho-ß-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1µM XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, ß-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-ß-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of ß-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-ß-catenin. Although XAV-939 does not downregulate active ß-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

Subclinical Endocarditis Might be a Hidden Trigger of Early Prosthetic Valve Calcification: A Histological Study.

OBJECTIVE: Despite various improvements in valve prosthetics, early valve deterioration still occurs, leading to prosthetic failure. Studying the early phase of this deterioration is quite difficult, as the prosthesis to be examined is almost always explanted only after extensive deterioration. The objective of this research is to study the pathology of early valve deterioration in an early stage in order to reveal the possible trigger of the process. METHODS: Three cusps of the same type of bovine pericardium valve prosthesis underwent comparative examination. Two cusps (cusps 1 and 2) were retrieved from a valve prosthesis explanted three months post-implantation, and the third cusp was from a non-implanted valve prosthesis and used as a reference cusp (ref. cusp). The examination included macroscopic examination, Non-linear Optical Microscopy using a multiphoton microscope, and histological examination with staining, using Hematoxylin and Eosin, Movat Pentachrome stain, Von-Kossa stain, and Alizirin-Red stain. Parallel sections were decalcified using Osteosoft® solution prior to Von-Kossa and Alizirin-Red staining to exclude false positive results. RESULTS: Macroscopically, cusp 1 showed early deterioration, and cusp 2 showed endocarditic vegetations. Histologically, cusp 1 showed calcifications in acellular deposits on the surface of the cusp, with pathological signs of subacute/healed endocarditis and intact cusp tissue. The examination did not show calcifications of the cellular remnants within the valve tissue. Cusp 2 showed florid endocarditis, with microscopic destruction of the valve tissue. CONCLUSION: Early prosthetic valve deterioration can exist as early as three months post-implantation. Subacute or subclinical endocarditis can be the cause for early valve calcification and deterioration.

Movat Pentachrom stain reveals unexpected high osteogenesis rate in aortic valves.

BACKGROUND AND AIM OF THE STUDY: Aortic valve (AV) stenosis is the most common valvular heart disease with an incidence of 3% for people ≥ 65years in the industrialized world with indication for a surgical or transcatheter valve replacement. Researchers suppose osteogenic processes as key mechanisms in calcific aortic valve stenosis. Recently, Torre et al. published impressive histological analyses and detected osseous and/or chondromatous metaplasia in 15.6% of 6685 native calcified aortic valves. Therefore one HE section per valve originated from the area with the greatest extent of calcification was analyzed. Aim of our experimental setup was to identify regions of neo-osteogenesis and to determine the rate of specimens with active mineralization in human aortic valve tissue by Movat Pentachrom staining of sections of lager tissue segments. METHODS: Operational replaced aortic valves of 35 patients, 15 female and 20 male with an average age of 66.2 years were formalin fixed and decalcified using Osteosoft®-solution. Tissue samples were cut and 2µm specimens were stained with Movat Pentachrom to visualize osteogenic regions. Instead of screening a large number of sections, tissue samples were cut up to five times with at least 100µm space each if no region of osseous and/or chondromatous metaplasia was visible. RESULTS/CONCLUSIONS: Using this setup, a region of osseous metaplasia was detected in 25 (71.4%) of 35 samples analyzed. In some cases, these regions were small sized and only visible due to the bright color of Movat Pentachrom stain. This leads to the suggestion that a higher rate of calcified aortic valve samples would be classified as cusps with areas of neo-osteogenesis after staining with Movat Pentachrom stain and by the systematic analysis of larger parts of the tissue blocks.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

Aldosterone Synthase Inhibition Improves Glucose Tolerance in Zucker Diabetic Fatty (ZDF) Rats.

Plasma aldosterone is elevated in type 2 diabetes and obesity in experimental and clinical studies and can act to inhibit both glucose-stimulated insulin secretion by the ß-cell and insulin signaling. Currently mineralocorticoid receptor antagonism is the best characterized treatment to ameliorate aldosterone-mediated effects. A second alternative is inhibition of aldosterone synthase, an approach with protective effects on end-organ damage in heart or kidney in animal models. The effect of aldosterone synthase inhibition on metabolic parameters in type 2 diabetes is not known. Therefore, male Zucker diabetic fatty (ZDF) rats were treated for 11 weeks with the aldosterone synthase inhibitor FAD286, beginning at 7 weeks of age. Results were compared with the mineralocorticoid receptor antagonist eplerenone. Plasma aldosterone was abolished by FAD286 and elevated more than 9-fold by eplerenone. The area under the curve calculated from an oral glucose tolerance test (OGTT) was lower and overall insulin response during OGTT was increased by FAD286. In contrast, eplerenone elevated blood glucose levels and blunted insulin secretion during the OGTT. Fasting glucose was lowered and fasting insulin was increased by FAD286 in the prediabetic state. Glycated hemoglobin was lowered by FAD286, whereas eplerenone showed no effect. We conclude that aldosterone synthase inhibition, in contrast to mineralocorticoid receptor antagonism, has the potential for beneficial effects on metabolic parameters in type 2 diabetes.

Heart valve stenosis in laser spotlights: insights into a complex disease.

Degenerative heart valve disease is a life-threatening disease affecting about 3% of the population over 65 years. Up to date, cardiac surgery with heart valve replacement is the only available therapy. The disease is characterized by degenerative disorganization of the heart valve structure and alterations in the residing cell populations. Causes and mechanisms of disease genesis are still not fully understood and until now pharmacological therapies are not available. Thus there is enormous interest in new technologies that enable a better characterization of structure and composition of diseased valves. Currently most research techniques demand for extensive processing of extracted valve material. We present a novel approach combining coherent anti-Stokes Raman scattering, endogenous two-photon excited fluorescence and second harmonic generation. Cusp constituents can be examined simultaneously, three-dimensionally and without extensive manipulation of the sample enabling impressive insights into a complex disease.

Reversal of myofibroblastic activation by polyunsaturated fatty acids in valvular interstitial cells from aortic valves. Role of RhoA/G-actin/MRTF signalling.

Valvular interstitial cells (VICs), the fibroblast-like cellular constituents of aortic heart valves, maintain structural integrity of valve tissue. Activation into contractile myofibroblasts occurs under pathological situations and under standard cell culture conditions of isolated VICs. Reversal of this phenotype switch would be of major importance in respect to fibrotic valve diseases. In this investigation, we found that exogenous polyunsaturated fatty acids (PUFAs) decreased contractility and expression of myofibroblastic markers like α-smooth muscle actin (αSMA) in cultured VICs from porcine aortic valves. The most active PUFAs, docosahexaenoic acid (DHA) and arachidonic acid (AA) reduced the level of active RhoA and increased the G/F-actin ratio. The G-actin-regulated nuclear translocation of myocardin-related transcription factors (MRTFs), co-activators of serum response factor, was also reduced by DHA and AA. The same effects were observed after blocking RhoA directly with C3 transferase. In addition, increased contractility after induction of actin polymerisation with jasplakinolide and concomitant expression of αSMA were ameliorated by active PUFAs. Furthermore, reduced αSMA expression under PUFA exposure was observed in valve tissue explants demonstrating physiological relevance. In conclusion, RhoA/G-actin/MRTF signalling is operative in VICs, and this pathway can be partially blocked by certain PUFAs whereby the activation into the myofibroblastic phenotype is reversed.