Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.

A time window for rescuing dying retinal ganglion cells.

BACKGROUND: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). METHODS: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca2+ were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy. RESULTS: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca2+ was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release. CONCLUSIONS: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.

Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures.

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

Rapid Internalization and Nuclear Translocation of CCL5 and CXCL4 in Endothelial Cells.

The chemokines CCL5 and CXCL4 are deposited by platelets onto endothelial cells, inducing monocyte arrest. Here, the fate of CCL5 and CXCL4 after endothelial deposition was investigated. Human umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were incubated with CCL5 or CXCL4 for up to 120 min, and chemokine uptake was analyzed by microscopy and by ELISA. Intracellular calcium signaling was visualized upon chemokine treatment, and monocyte arrest was evaluated under laminar flow. Whereas CXCL4 remained partly on the cell surface, all of the CCL5 was internalized into endothelial cells. Endocytosis of CCL5 and CXCL4 was shown as a rapid and active process that primarily depended on dynamin, clathrin, and G protein-coupled receptors (GPCRs), but not on surface proteoglycans. Intracellular calcium signals were increased after chemokine treatment. Confocal microscopy and ELISA measurements in cell organelle fractions indicated that both chemokines accumulated in the nucleus. Internalization did not affect leukocyte arrest, as pretreatment of chemokines and subsequent washing did not alter monocyte adhesion to endothelial cells. Endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings expand our knowledge of how chemokines attract leukocytes to sites of inflammation.

A collagen-binding protein enables molecular imaging of kidney fibrosis in vivo.

Pathological deposition of collagen is a hallmark of kidney fibrosis. To illustrate this process we employed multimodal optical imaging to visualize and quantify collagen deposition in murine models of kidney fibrosis (ischemia-reperfusion or unilateral ureteral obstruction) using the collagen-binding adhesion protein CNA35. For in vivo imaging, we used hybrid computed tomography-fluorescence molecular tomography and CNA35 labeled with the near-infrared fluorophore Cy7. Upon intravenous injection, CNA35-Cy7 accumulation was significantly higher in fibrotic compared to non-fibrotic kidneys. This difference was not detected for a non-specific scrambled version of CNA35-Cy7. Ex vivo, on kidney sections of mice and patients with renal fibrosis, CNA35-FITC co-localized with fibrotic collagen type I and III, but not with the basement membrane collagen type IV. Following intravenous injection, CNA35-FITC bound to both interstitial and perivascular fibrotic areas. In line with this perivascular accumulation, we observed significant perivascular fibrosis in the mouse models and in biopsy sections from patients with chronic kidney disease using computer-based morphometry quantification. Thus, molecular imaging of collagen using CNA35 enabled specific non-invasive quantification of kidney fibrosis. Collagen imaging revealed significant perivascular fibrosis as a consistent component next to the more commonly assessed interstitial fibrosis. Our results lay the basis for further probe and protocol optimization towards the clinical translation of molecular imaging of kidney fibrosis.

Molecular Imaging in Oncology: Advanced Microscopy Techniques.

Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many microscopy techniques exist that can offer this information, also for malignant tissues and therapeutic approaches. In this overview, we discuss the various advanced optical microscopy techniques and their applications in oncological research. After a short introduction in Sect. 16.1, we continue in Sect. 16.2 with a discussion on fluorescent labelling strategies, followed in Sect. 16.3 by an in-depth description of confocal, light-sheet, two-photon, and super-resolution microscopy. We end in Sect. 16.4 with a focus on the applications, specifically in oncology.

Biomass Pretreatment and Enzymatic Hydrolysis Dynamics Analysis Based on Particle Size Imaging.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass' cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 µm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

Decoration of intramyocellular lipid droplets with PLIN5 modulates fasting-induced insulin resistance and lipotoxicity in humans.

AIMS/HYPOTHESIS: In contrast to insulin-resistant individuals, insulin-sensitive athletes possess high intramyocellular lipid content (IMCL), good mitochondrial function and high perilipin 5 (PLIN5) levels, suggesting a role for PLIN5 in benign IMCL storage. We hypothesised a role for PLIN5 in modulating fasting-mediated insulin resistance. METHODS: Twelve men were fasted for 60 h, before and after which muscle biopsies were taken and stained for lipid droplets (LDs), PLIN5 and laminin. Confocal microscopy images were analysed for LD size, number, PLIN5 association and subcellular distribution. RESULTS: Fasting elevated IMCL content 2.8-fold and reduced insulin sensitivity (by 55%). Individuals with the most prominent increase in IMCL showed the least reduction in insulin sensitivity (r = 0.657; p = 0.028) and mitochondrial function (r = 0.896; p = 0.006). During fasting, PLIN5 gene expression or PLIN5 protein content in muscle homogenates was unaffected, microscopy analyses revealed that the fraction of PLIN5 associated with LDs (PLIN5+) increased significantly (+26%) upon fasting, suggesting PLIN5 redistribution. The significant increase in LD number (+23%) and size (+23%) upon fasting was entirely accounted for by PLIN5+ LDs, not by LDs devoid of PLIN5. Also the association between IMCL storage capacity and insulin resistance and mitochondrial dysfunction was only apparent for PLIN5+ LDs. CONCLUSIONS/INTERPRETATION: Fasting results in subcellular redistribution of PLIN5 and promotes the capacity to store excess fat in larger and more numerous PLIN5-decorated LDs. This associates with blunting of fasting-induced insulin resistance and mitochondrial dysfunction, suggesting a role for PLIN5 in the modulation of fasting-mediated lipotoxicity. TRIAL REGISTRATION: trialregister.nl NTR 2042.

Calcium signaling recruits substrate transporters GLUT4 and CD36 to the sarcolemma without increasing cardiac substrate uptake.

Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca(2+)/calmodulin-activated kinase kinase-ß (CaMKKß) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca(2+)]i stimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca(2+) signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca(2+) and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca(2+) signaling affected neither AMPK-Thr(172) phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca(2+) signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca(2+) signaling and AMPK activators, Ca(2+) signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca(2+) signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKß/CaMKs. Ca(2+)-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca(2+)-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca(2+) signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation.

Ex vivo and in vivo administration of fluorescent CNA35 specifically marks cardiac fibrosis.

Cardiac fibrosis is a major hallmark of cardiac diseases. For evaluation of cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. Our aim was to evaluate CNA35, a protein known to specifically bind to collagen, as a specific marker of cardiac fibrosis. Fluorescently labeled CNA35 was applied ex vivo on tissue sections of fibrotic rat, mouse, and canine myocardium. After quantification of CNA35, sections were examined with picrosirius red (PSR) and compared to CNA35. Furthermore, fluorescently labeled CNA35 was administered in vivo in mice. Hearts were isolated, and CNA35 labeling was examined in tissue sections. Serial sections were histologically examined with PSR. Ex vivo application of CNA35 showed specific binding to collagen and a high correlation with PSR (Pearson r  =  .86 for mice/rats and r  =  .98 for canine; both p < .001). After in vivo administration, CNA35 labeling was observed around individual cardiomyocytes, indicating its ability to penetrate cardiac endothelium. High correlation was observed between CNA35 and PSR (r  =  .91, p < .001). CNA35 specifically binds to cardiac collagen and can cross the endothelial barrier. Therefore, labeled CNA35 is useful to specifically detect collagen both ex vivo and in vivo and potentially can be converted to a noninvasive method to detect cardiac fibrosis.

Murine bladder imaging by 2-photon microscopy: an experimental study of morphology.

PURPOSE: We developed 2-photon laser scanning microscopy analysis of the native murine bladder. MATERIALS AND METHODS: Bladder tissue from wild-type mice was imaged by 2-photon laser scanning microscopy autofluorescence and second harmonic generation microscopy. Bladder wall layers and structures were analyzed using differences in color, size, shape and morphology. RESULTS: Autofluorescence of the urothelium, nerve structures and muscles was visible in the green spectral channel due to autofluorochromes such as NAD(P)H and elastin. Second harmonic generation of collagen was seen in the blue spectral channel. Imaging from the mucosal side revealed umbrella cells at 0 and 30 µm, of which the high cellular NAD(P)H content allows autofluorescence detection. Below that a network-like connective tissue layer was visualized up to 50 µm that contained vessels with a diameter of 10 to 40 µm and nerves with a diameter of 1 to 6 µm. Imaging from the adventitial side revealed a radiant collagen layer covered with nerves and macrophages at 0 to 20 µm. Below at 20 to 25 µm we visualized a thick muscle layer containing elastic fibers and macrophages. Findings were also represented in 3-dimensional reconstructions, providing information on structure localization, orientation and interconnection. CONCLUSIONS: Two-photon laser scanning microscopy imaging using autofluorescence of the murine bladder is a promising technique to provide new insight into structures and morphology. It opens avenues to identify structural changes in bladder pathology.

Plaque-associated vasa vasorum in aged apolipoprotein E-deficient mice exhibit proatherogenic functional features in vivo.

OBJECTIVE: Neovascularization of human atherosclerotic plaques is implicated in plaque progression and destabilization, although its functional implications are yet unresolved. Here, we aimed to elucidate functional and morphological properties of plaque microvessels in mice in vivo. METHODS AND RESULTS: Atherosclerotic carotid arteries from aged (>40 weeks) apolipoprotein E-deficient mice were imaged in vivo using multiphoton laser scanning microscopy. Two distinct groups of vasa vasorum microvessels were observed at sites of atherosclerosis development (median diameters of 18.5 and 5.9 µm, respectively), whereas microvessels within the plaque could only rarely be found. In vivo imaging showed ongoing angiogenic activity and injection of fluorescein isothiocyanate-dextran confirmed active perfusion. Plaque vasa vasorum showed increased microvascular leakage, combined with a loss of endothelial glycocalyx. Mean blood flow velocity in plaque-associated vasa vasorum was reduced by ±50% compared with diameter-matched control capillaries, whereas mean blood flow was reduced 8-fold. Leukocyte adhesion and extravasation were increased 6-fold in vasa vasorum versus control capillaries. CONCLUSIONS: Using a novel in vivo functional imaging strategy, we showed that plaque-associated vasa vasorum were angiogenically active and, albeit poorly, perfused. Moreover, plaque-associated vasa vasorum showed increased permeability, reduced blood flow, and increased leukocyte adhesion and extravasation (ie, characteristics that could contribute to plaque progression and destabilization).

Multiphoton Imaging of Maturation in Tissue Engineering.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

CD36 inhibition prevents lipid accumulation and contractile dysfunction in rat cardiomyocytes.

An increased cardiac fatty acid supply and increased sarcolemmal presence of the long-chain fatty acid transporter CD36 are associated with and contribute to impaired cardiac insulin sensitivity and function. In the present study we aimed at preventing the development of insulin resistance and contractile dysfunction in cardiomyocytes by blocking CD36-mediated palmitate uptake. Insulin resistance and contractile dysfunction were induced in primary cardiomyocytes by 48 h incubation in media containing either 100 nM insulin (high insulin; HI) or 200 µM palmitate (high palmitate; HP). Under both culture conditions, insulin-stimulated glucose uptake and Akt phosphorylation were abrogated or markedly reduced. Furthermore, cardiomyocytes cultured in each medium displayed elevated sarcolemmal CD36 content, increased basal palmitate uptake, lipid accumulation and decreased sarcomere shortening. Immunochemical CD36 inhibition enhanced basal glucose uptake and prevented elevated basal palmitate uptake, triacylglycerol accumulation and contractile dysfunction in cardiomyocytes cultured in either medium. Additionally, CD36 inhibition prevented loss of insulin signalling in cells cultured in HP, but not in HI medium. In conclusion, CD36 inhibition prevents lipid accumulation and lipid-induced contractile dysfunction in cardiomyocytes, but probably independently of effects on insulin signalling. Nonetheless, pharmacological CD36 inhibition may be considered as a treatment strategy to counteract impaired functioning of the lipid-loaded heart.

Two-Photon Endoscopy: State of the Art and Perspectives.

In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. Several groups have recently presented their development towards the goal of producing user friendly plug and play system, which could be used in biological research and, potentially, clinical applications. We first present the technological challenges, prerequisites, and solutions in two-photon endoscopic systems. Secondly, we focus on the applications already found in literature. These applications mostly serve as a quality check of the built system, but do not answer a specific biomedical research question. Therefore, in the last part, we will describe our vision on the enormous potential applicability of adult two-photon endoscopic systems in biological and clinical research. We will thus bring forward the concept that two-photon endoscopy is a sine qua non in bringing this technique to the forefront in clinical applications.

Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release.

Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca2+ and reactive oxygen species, cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing, PS exposure and Cyto.C release into the cytosol. Removing EtOH at predetermined time points revealed that all phenomena except Cyto.C release occurred in a phase of neuronal cell death in which full recovery to a neurite-bearing cell was still possible. Our findings underscore a strategy of treating chronic neurodegenerative diseases by removing stressors from neurons and harnessing intracellular targets that delay or prevent trespassing the point of no return.

Blocking of frizzled signaling with a homologous peptide fragment of wnt3a/wnt5a reduces infarct expansion and prevents the development of heart failure after myocardial infarction.

BACKGROUND: The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. METHODS AND RESULTS: Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. CONCLUSIONS: Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.

The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.